We would like to thank Lindsey W. Plasschaert and Justin Sui for their substantial contributions to the early development of this protocol. The authors are grateful to the fly community for their generosity with reagents, and particularly to Ruth Lehmann and Benjamin Lin for their gift of the nos5&#8217;-Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#39; line prior to its publication. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was supported by NIH RO1 GM060804, R33AG04791503 and R35GM136270 (S.D) as well as training grants T32GM007229 (B.W.) and F32GM125123 (L.A.).

1. Day-before-dissection preparation
<ol>
	<li>Electrolytically sharpen a tungsten needle24 so that the resulting diameter is approximately 0.03 mm. Adjust the voltage supplied to approximately 14 V, and use 3.3 M NaOH. Sharpening should take no more than 1 or 2 min. 
	CAUTION: NaOH is highly corrosive and will cause burns upon contact with skin. Wear gloves and goggles while handling, and work inside a fume hood. 
	NOTE: After use, store NaOH in a polypropylene Falcon tube.</li>
	<li>...

<ol>
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W., Sui, J., DiNardo, S. <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+reveals+hub+cell+assembly+and+compaction+dynamics+during+morphogenesis+of+the+Drosophila+testis+niche.">Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche.</a> Developmental Biology. 446, 102-118 (2019).</li><li>Rebecca Sheng, X., Matunis, E. <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+the+Drosophila+spermatogonial+stem+cell+niche+reveals+novel+mechanisms+regulating+germline+stem+cell+output.">Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.</a> Development. 138, 3367-3376 (2011).</li><li>Aboim, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developpement+embryonnaire+et+post-embryonnaire+des+gonades+normales+et+agametiques+de+Drosophila+melanogaster.">Developpement embryonnaire et post-embryonnaire des gonades normales et agametiques de Drosophila melanogaster.</a> Revue Suisse De Zoologie. 52, 53 (1945).</li><li>Sano, H., 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+Drosophila+actin+regulator+ENABLED+regulates+cell+shape+and+orientation+during+gonad+morphogenesis.">The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.</a> PLoS One. 7, (2012).</li><li>Clark, I. B. N., Jarman, A. P., Finnegan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+gonad+formation+reveals+roles+for+Six4+in+regulating+germline+and+somatic+cell+migration.">Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration.</a> BMC Devlopmental Biology. 7, 1-9 (2007).</li><li>Sheng, X. R., Brawley, C. M., Matunis, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiating+spermatogonia+outcompete+somatic+stem+cells+for+niche+occupancy+in+the+Drosophila+testis.">Dedifferentiating spermatogonia outcompete somatic stem cells for niche occupancy in the Drosophila testis.</a> Cell Stem Cell. 5, 191-203 (2009).</li><li>Tanentzapf, G., Devenport, D., Godt, D., Brown, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Europe+PMC+Funders+Group+integrin-dependent+anchoring+of+a+stem+cell+niche.">Europe PMC Funders Group integrin-dependent anchoring of a stem cell niche.</a> Nature Cell Biology. 9, 1413-1418 (2007).</li><li>Brady, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+making+very+fine,+durable+dissecting+needles+by+sharpening+tungsten+wire+electrolytically+a+motorized+molluscicide+dispenser.">A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically a motorized molluscicide dispenser.</a> Bulletin of the World Health Organization. , 105-106 (1960).</li><li>Featherstone, D. 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The authors have nothing to disclose.

During gonadogenesis, the embryonic gonad, and particularly the stem cell niche within the male gonad15, undergoes rapid morphological changes. Developmental mechanisms that underlie these dynamic changes are best understood through live-imaging techniques. However, at embryonic Stage 17, in vivo imaging of the gonad is rendered impossible by the onset of large-scale muscle contractions17. With this protocol, we provide a successful alternative: dissection of the g...

The Drosophila melanogaster testis has served as a paradigm for our understanding of many dynamic cellular processes. Studies of this model have shed light on stem cell division regulation1,2,3, germ cell development4,5, piRNA biology6,7,8, and niche-stem cell signaling events9,10,11,12,13. This model is advantageous because it is genetically tractable14,15 and is one of the few where we can live-image stem cells in their natural environment3,16,17,18. However, live-imaging of this model has been limited to adult tissue and early embryonic stages, leaving a gap in our knowledge of gonadal dynamics in the late embryo, the precise stage when the niche is first forming and beginning to function.
The late stage embryonic gonad is a sphere, consisting of somatic niche cells at the anterior, and germ cells encysted by somatic gonadal cells throughout more posterior regions19. This organ can be imaged live in vivo up until early embryonic Stage 1717,20,21. Further imaging is prevented due to initiation of large-scale muscle contractions. These contractions are so severe that they push the gonad out of the imaging frame, and such movement cannot be corrected with imaging software. Our lab is interested in unveiling the mechanisms of niche formation, which occurs during this elusive period for live-imaging. Therefore, we generated an ex vivo approach to live image the gonad starting at embryonic Stage 16, facilitating the study of the cell dynamics during this crucial period of gonad development. Previous work from our lab shows that this ex vivo imaging faithfully recapitulates in vivo gonad development17. This technique is the first and only of its kind for the Drosophila embryonic gonad.
Here, we present the dissection protocol required for ex vivo live-imaging of the gonad during late embryonic stages. This protocol can be combined with pharmacological treatments, or transgenic manipulation of specific cell lineages within the gonad. Using this technique, we have successfully imaged the steps of stem cell niche formation17. This imaging approach is thus instrumental for the field of stem cell biology, as it will enable visualization of the initial stages of niche formation in real time within its natural environment15,17. While this method is beneficial for the field of stem cell biology, it is additionally applicable for visualizing any dynamic processes occurring in the gonad during this developmental timepoint, including cellular rearrangements22, cell adhesion2,12,23, and cell migration23. This dissection protocol will thus enhance our understanding of many fundamental cell biological processes.

<table>
	<tbody>
		<tr>
			<td>Alfa Aesar&#160;Tungsten wire</td>
			<td>Fisher Scientific</td>
			<td>AA10408G6</td>
			<td>0.25mm (0.01 in.) dia., 99.95% (metals basis)</td>
		</tr>
		<tr>
			<td>D. melanogaster: His2Av::mRFP1</td>
			<td>Bloomington Drosophila Stock Center (BDSC)</td>
			<td>FBtp0056035</td>
			<td>Schuh, Lehner, &#38; Heidmann, Current Biology, 2007</td>
		</tr>
		<tr>
			<td>D. melanogaster: nos-lifeact::tdtomato</td>
			<td>Gift from Ruth Lehmann Lab</td>
			<td></td>
			<td>Lin, Luo, &#38; Lehmann, Nature Communications, 2020: nos5&#39;- Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#39;</td>
		</tr>
		<tr>
			<td>D. melanogaster: P-Dsix4-eGFP::Moesin</td>
			<td></td>
			<td>FBtp0083398</td>
			<td>Sano et al., PLoS One, 2012</td>
		</tr>
		<tr>
			<td>Diamond-tipped knife</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>MetaMorph Microscopy Automation and Image Analysis Software v7.8.4.0</td>
		</tr>
		<tr>
			<td>Insulin, bovine</td>
			<td>Sigma</td>
			<td>l0516</td>
			<td>Store aliquots at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fisher Scientific</td>
			<td>08-955</td>
			<td></td>
		</tr>
		<tr>
			<td>Nytex basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td></td>
			<td></td>
			<td>2 mM MgCl2, 2 mM CaCl2, 130 mM NaCl, 5mM KCl, 36 mM Sucrose, 5mM Hepe&#8217;s Buffer; adjusted with NaOH until pH of 7.3 is achieved</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Media</td>
			<td>GIBCO</td>
			<td>21720-024</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We illustrate preparation of the imaging dish in Figure 1, as described in &#8220;Day-of-dissection preparation.&#8221; These methods should ultimately result in well-hydrated embryos adhered to a cover slip strip, which is temporarily fixed to the bottom of the dish and submerged in Ringer&#8217;s solution (Figure 1F). A diamond-tipped knife allows one to cleanly slice a 22 x 22 mm cover slip into three to four smaller strips (Figure 1A</st...

Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

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.

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, ...

Cell dynamics are most easily understood with live imaging. This protocol enables live imaging of the developmental stage of gonadogenesis that was previously inaccessible to visualization. The fine dissections necessary to isolate the gonad require delicate and precise manipulations under high magnification.Only a visual demonstration can convey how to execute each step. Someone new to this technique might struggle with rapid timing and delicate tissue manipulations required at certain steps. We recommend practicing sections of this protocol separately while learning.Begin by cutting a 22-by-22-millimeter cover slip into four equally sized strips using a diamond-tipped knife. Pick up one strip using forceps, and spread 30 microliters of heptane glue solution on both sides of the strip. Tilt the strip at various angles to achieve an even layer of glue residue as the heptane evaporates.Store the strip in an empty cover slip box. Set a slanted but upright position to minimize contact with the box and to maintain its stickiness. Then close the box.Transfer aged dechorionated embryos to a small watch glass filled with 500 to 750 microliters of heptane. Draw the embryos into the narrow portion of the micro pipette and slowly aggregate them near the inner tip of the pipette before gently pipetting them onto the slide. Twist the corner of a tissue wipe into a fine tip and wick away heptane from the embryos on the slide, making it easier to capture them on a glue-covered strip.Pick up a glue-covered strip using forceps and gently touch it to the embryos. Place the strip in the imaging dish, just outside the Poly-D-Lysine-coated center with embryo side up. Press the strip bound to the dish to ensure that it is fixed in place.Immediately flood the dish with two to 3.5 milliliters of Ringer's solution to submerge the embryos and prevent them from drying. During the dissection, tissue debris may stick to the dissecting needle. To get rid of this debris, periodically raise the needle just above the surface of the Ringer's solution.Select stage 16 embryos with three gut constrictions that create four stacked gut segments. To begin devitellinization, pierce the selected embryo at the anterior end with a tungsten needle and peel the vitelline membrane off the embryo. Hook the needle through the embryo in a region far from the gonads and transfer the embryo to the polylysine-coated region of the cover slip.Drag it against the bottom until it adheres. Similarly, arrange all the devitellinized embryos in a row along the top of the cover slip, leaving plenty of space below. Filet the embryo to expose its interior and slice through the embryo from its center by moving the needle posteriorly between the gonads.Tease out some internal tissue to reveal the gonad and allow it to adhere to the cover slip. Using a needle, slice a piece of tissue, including the gonad, and separate it from the remaining carcass. Then, draw this tissue to a fresh region of the coated cover slip to adhere it to the dish.Remove as much of the surrounding tissue from the gonad as possible by carefully guiding away the extraneous tissue and adhering the gonad to a fresh sticky region of the dish. Make sure not to touch the goad directly to avoid damaging it. Following dissection, add registration marks to the outside of the dissecting dish to help with locating gonads at the confocal microscope later.Remove the glue-coated strip from the dish by gently inserting the bottom prong of a pair of forceps under the strip, clasping the forceps, and then tilting the strip upwards to free it from the dish minimizing disturbance of the adhered gonads. Place the imaging dish in the stage holder using the registration marks to place the dish in the same approximate orientation as during dissection. Use Brightfield microscopy and a lower power objective to identify and focus on any piece of tissue adhered to the cover slip.Switch the IP settings to reveal fluorescence, and use the binocular eye pieces to systemically scan the dish, marking the position of each gonad within the imaging software. Gently remove the entire stage holder assembly with the imaging dish still in its holder and place the assembly on the work bench. Remove some of the Ringer's solution with a P1000 micropipette from the inside upper ledge of the imaging dish.Then remove approximately 50 to 100 microliters of the solution from under the surface using a P200 micropipette, making sure not to remove all solution. Add approximately 200 microliters of imaging media containing insulin under the surface of the remaining Ringer's solution. Add the remaining imaging media to the dish, starting at the outermost rim of the upper ledge.While pipetting, move toward the central dome of the fluid and eventually merge the tube by brushing the pipette tip across both. Switch the microscope to a higher power objective and apply proper immersion fluid. Then replace the stage holder assembly.Adjust the focus to the marked gonad position and begin imaging. Bright field visualization of an isolated gonad on a confocal microscope reveals a dark shadow surrounding the gonad periphery, which is the extracellular matrix that maintains the integrity of gonads during imaging. An example of a healthy well-cultured gonad expressing labeled and somatic gonadal cells and histone markers is shown here.If the gonads are not sufficiently hydrated during imaging, the tissue shrivels. A gonad whose extracellular matrix is damaged during dissection has a compromised boundary, which is evident by the presence of gonad-specific cells outside of the confines of the gonad. Prior to compaction, the forming stem cell niche in the X-VIVO cultured gonad initially appears as a loose aggregate of somatic cells at the gonad anterior.The niche cells are surrounded by germline stem cells. The niche aggregate initially exhibits an irregular boundary. Throughout the course of imaging, the individual niche cells rearrange their positions such that the niche aggregate acquires smoother borders, resulting in a decrease of the niche area.When attempting this protocol it is important to work quickly when transferring embryos to a coated dish and to be delicate when dissecting the gonad away from the surrounding tissue. X-VIVO culture can be combined with both genetic and pharmacological manipulations, allowing for multifaceted approaches to investigate events occurring during male or female gonadogenesis. This technique made it possible for us to visualize how the stem cell niche acquires its final architecture.Consequently, we can now form and test hypotheses about underlying cell dynamics that drive its morphogenesis.

dissection-and-live-imaging-of-the-late-embryonic-drosophila-gonad

Research

JoVE Journal

Developmental Biology

3.1K Görüntüleme.  University of Pennsylvania. Cell dynamics are most easily understood with live imaging. This protocol enables live imaging of the developmental stage of gonadogenesis that was previously inaccessible to visualization. The fine dissections necessary to isolate the gonad require delicate and precise manipulations under high magnification.Only a visual demonstration can convey how to execute each step. Someone new to this technique might struggle with rapid timing and delicate tissue manipulations required at certai...