We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

1. Dissection of Ovarioles
<ol>
	<li>Transfer about fifteen 2-4-day-old female flies and a few males to a fresh fly food vial with added active dry yeast. Use cotton as a plug for the vials, and add a few drops of water to the cotton to keep the humidity high.</li>
	<li>Place vial in a 25 &#176;C incubator for 14-16 hr to maximize the number of stage 8-10 egg chambers. 
	NOTE: Incubation time varies depending on temperature and desired stage.</li>
	<li>Prepare dissection media, 0.1M KPO4 and NP40 soluti...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+morphogenetic+repertoire:+perspectives+from+the+Drosophila+egg.">Expanding the morphogenetic repertoire: perspectives from the Drosophila egg.</a> Dev Cell. 22 (1), 12-23 (2012).</li><li>Montell, D. J., Yoon, W. H., Starz-Gaiano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+choreography:+mechanisms+orchestrating+the+collective+movement+of+border+cells.">Group choreography: mechanisms orchestrating the collective movement of border cells.</a> Nat Rev Mol Cell Biol. 13 (10), 631-645 (2012).</li><li>Spradling, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border-cell+migration:+the+race+is+on.">Border-cell migration: the race is on.</a> Nat Rev Mol Cell Biol. 4 (1), 13-24 (2003).</li><li>Silver, D., Geisbrecht, E., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+JAK/STAT+signaling+throughout+border+cell+migration+in+Drosophila.">Requirement for JAK/STAT signaling throughout border cell migration in Drosophila.</a> Development. 132 (15), 3483-3492 (2005).</li><li>Ghiglione, C., 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+cytokine+receptor+Domeless+controls+border+cell+migration+and+epithelial+polarization+during+oogenesis.">The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis.</a> Development. 129 (23), 5437-5447 (2002).</li><li>Denef, N., Sch&#252;pbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning:+JAK-STAT+signalling+in+the+Drosophila+follicular+epithelium.">Patterning: JAK-STAT signalling in the Drosophila follicular epithelium.</a> Curr Biol. 13 (10), R388-R390 (2003).</li><li>Beccari, S., Teixeira, L., R&#248;rth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK/STAT+pathway+is+required+for+border+cell+migration+during+Drosophila+oogenesis.">The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis.</a> Mech Dev. 111 (1-2), 115-123 (2002).</li><li>Montell, D., Rorth, P., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slow+border+cells,+a+locus+required+for+a+developmentally+regulated+cell+migration+during+oogenesis,+encodes+Drosophila+C/EBP.">slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP.</a> Cell. 71 (1), 51-62 (1992).</li><li>Xi, R., McGregor, J., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gradient+of+JAK+pathway+activity+patterns+the+anterior-posterior+axis+of+the+follicular+epithelium.">A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium.</a> Dev Cell. 4 (2), 167-177 (2003).</li><li>Toomre, D., Bewersdorf, 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+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annu Rev Cell Dev Biol. 26, 285-314 (2010).</li><li>McDonald, J., Pinheiro, E., Kadlec, L., Schupbach, T., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+EGFR+ligands+participate+in+guiding+migrating+border+cells.">Multiple EGFR ligands participate in guiding migrating border cells.</a> Dev Biol. 296 (1), 94-103 (2006).</li><li>Van de Bor, V., Zimniak, G., C&#233;r&#233;zo, D., Schaub, S., Noselli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+localisation+of+cytokine+mRNA+is+essential+for+JAK/STAT+activation+during+cell+invasiveness.">Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness.</a> Development. 138 (7), 1383-1393 (2011).</li><li>Hayashi, Y., 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=Glypicans+regulate+JAK/STAT+signaling+and+distribution+of+the+Unpaired+morphogen.">Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen.</a> Development. 139 (22), 4162-4171 (2012).</li><li>Airoldi, S. 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Here we describe a method to mount and image small, developing egg chambers from a end-on perspective. Common techniques for imaging egg chambers are optimized for lateral views and primarily allow precise visualization of medio-lateral follicle cells when stained with fluorescent antibodies. The use of Z-stacks or 3-D reconstructions aids in viewing multiple focal planes, but is still inadequate for sub-cellular resolution of the poles of elliptical egg chambers (Figure 2D). While this can be overcome p...

Study of Drosophila melanogaster has provided great insights into the genetic regulation of a wide range of phenomena. In particular, there has been extensive research on egg development, because oogenesis provides a tractable way to investigate many different developmental processes, including tissue patterning, cell polarity changes, cell cycle switching, and translational regulation1,2,3,4. One important morphogenic event during oogenesis is the specification, acquisition of motility, and migration of a set of cells called border cells (reviewed in 5). Since cell migration is a key feature of animal morphogenesis, and because the genetic regulation of this process is well-conserved, mechanisms determined in flies are likely to be important in other contexts. Thus, we are investigating the molecular control of border cell migration. For our studies, we have developed a new method to observe the anterior poles of developing eggs, where border cells arise, to examine how they develop in detail.
Within the ovary, egg chambers at various developmental stages exist along chains called ovarioles, which are encased in a thin sheath (Figure 1A). Each egg chamber will go on to form one egg. During oogenesis, several different cell types must develop in a coordinated manner. The D. melanogaster egg chamber consists of 16 germ-line cells, including one oocyte, surrounded by a single-layer follicular epithelium6. A small number of specialized cells, called polar cells, arise at the anterior and posterior poles of the epithelium. The 6-8 border cells originate in the anterior epithelium of the egg chamber, induced by the polar cells5,7. In mid-oogenesis (stage 9), the border cells detach from their neighbors and migrate between the nurse cells to reach the oocyte at the posterior of the egg chamber5,7. This movement must be accomplished while the border cells remain in a cluster surrounding two non-motile polar cells, making this a type of collective cell migration. Successful migration of the border cell cluster ensures proper development of the micropyle of the egg shell, which is necessary for fertilization.
The anterior polar cells instruct border cell fate by activating a signal transduction cascade. Polar cells secrete a cytokine, Unpaired (UPD), which binds to a transmembrane receptor, Domeless (DOME), on neighboring follicle cells during stage 8 of oocyte development8,9. The binding of UPD causes Janus tyrosine kinase (JAK) to phosphorylate the Signal Transducer and Activator of Transcription (STAT)8,10,11. STAT then moves to the nucleus to activate transcription. Slow Border Cells (SLBO) is a transcription factor that is a direct transcriptional target of STAT and is also required for border cell migration12. Lateral views of egg chambers indicate that STAT activity is regulated in a gradient across the anterior epithelium8,11,13. Follicle cells closest to the polar cells have the highest levels of activated STAT, thus they become border cells and invade the adjacent germ-line tissue.
To understand how the border cells are specified within and detach from the epithelium, we need to observe how the tissue is organized. If we view egg chambers from an anterior-on perspective, we would expect radial symmetry of STAT activity in the follicle cells surrounding the polar cells. An end-on view would also more accurately show differences of membrane proteins and cell-cell interfaces prior to and during detachment than comparing cells in different focal planes. Because egg chambers are oblong and attached to each other by stalk cells, they settle onto slides laterally, making it difficult to observe the anterior architecture. Thus, much information about the cells at the poles of the egg chamber has been inferred from lateral views. Although some information can be obtained through algorithmic 3-D reconstructions of optical sections, light scattering, photobleaching, and poorer limits of resolution in the Z-axis make this information less detailed and reliable in the absence of expensive techniques like super-resolution microscopy14. Other kinds of section-based imaging (for example, electron microscopy or microtome sectioning) require extensive manipulation of tissues, including dehydration, increasing the likelihood for artifacts. Thus, we developed a new method to image D. melanogaster egg chambers while upright. This method has already proven useful in elucidating how motile cells are fated (see Representative results and Manning et al, under review), and is likely to be more broadly valuable in studies of other aspects of oogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1027">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4&#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Glass culture tube</td>
			<td style="width:279px;">VWR</td>
			<td>47729-576</td>
			<td>14mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
			<td style="width:279px;">VWR</td>
			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerin Jelly&#160;</td>
			<td style="width:279px;">Electron Microsocpy Sciences</td>
			<td>17998-10</td>
			<td>Mounting media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerol</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB15760</td>
			<td>70% in PBS</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>For processing confocal Z-stacks</td>
		</tr>
	</tbody>
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upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

The upright imaging method allowed us to see directly the organization of cells in the anterior follicular epithelium at stage 8. A general marker for follicle cell fate, the Eyes Absent (EYA) protein, as well as the nuclear DNA marker DAPI, showed even expression across this field of cells, and demonstrated that all cells could be seen with similar staining intensities (Figure 2B&#8221;). Proteins regulated in response to the cytokine UPD, however, showed variable patterns and expression levels. Polar c...

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Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

7.5K 次观看.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.