Name: the utility of stage-specific mid-to-late drosophila follicle isolation
Uploaded: 2013-12-02T20:55:00
Description: Watch this Scientific Journal Video about The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation at JoVE.com

We would like to thank Thomas Lecuit (sqh-Utrophin::GFP line), the Bloomington Stock Center, and the Developmental Studies Hybridoma Bank for reagents. We further thank all members of the Tootle Lab for helpful discussions and critiques of the manuscript. Funding from the National Science Foundation MCB-1158527, and start-up funds from the Anatomy and Cell Biology Department, University of Iowa supported this work. National Institutes of Health Predoctoral Training Grant in Pharmacological Sciences T32GM067795 supported AJS. Data storage support was provided by the ICTS, which is funded through the CTSA supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1RR024979.

1. Preparing Drosophila Prior to Stage Isolation
<ol>
	<li>Make wet yeast paste by combining 50 g of active dry yeast with 90 ml&#160;of distilled water. Mix with a spatula to combine. Let the mixture stand for ~30 min before assessing consistency. The consistency should be just thick enough to adhere to the side of a fly vial but not run down the side. To achieve the desired consistency it may be necessary to add up to 10 ml&#160;of additional water or a small amount of dry yeast. Store in a covered container...

<ol>
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The Drosophila follicle is composed of only a small number of cell types, making it ideal for both morphologic and molecular analyses. Furthermore, due to the structure of the ovary, it is relatively easy to obtain large numbers of specific stages of follicle development with a common dissecting scope and minimal training. As each stage represents a short temporal window, stage isolation can provide significant molecular insights into the developmental processes occurring during that stage. For example, we have ...

Each Drosophila ovary is composed of ~16 ovarioles, or chains of sequentially maturing egg chambers or follicles. Each follicle is composed of a single oocyte, 15 germ line derived nurse or support cells, and ~650 somatic cells termed follicle cells (Figure 1A). Drosophila oogenesis is divided into 14 morphologically defined stages of development1. Each stage of follicle development is observed many times within a single fly,&#160;making it relatively easy to isolate a substantial number of stage-specific follicles.
The mid-to-late stages of oogenesis (Stages 10B-14) are particularly well suited for stage isolation (Figure 1). At Stage 10B (S10B), the follicle is fully elongated (i.e. its length is equal to that of a Stage 14 (S14) follicle, see Figure 1 and Figure 2H) and half the length of the follicle is composed of nurse cells while the other half is the oocyte (Figure 1C). At this stage the nurse cells undergo dramatic actin remodeling, strengthening the cortical actin and generating parallel bundles of actin filaments2. At the same time, a population of follicle cells, termed centripetal cells, migrate in between the nurse cells and the oocyte, and two dorsal groups of follicle cells become specified to undergo migration to form the dorsal appendages, tubular respiratory apparatuses for the embryo3. The nurse cells then contract (S11), squeezing their cytoplasmic contents into the oocyte in a process called nurse cell dumping, which provides the oocyte with the factors necessary for it to complete embryogenesis (Figure 1D). The nurse cells then undergo cell death (S12-S13)4, and the follicle cells secrete and pattern the eggshell5 (Figures 1E-G). Thus, the end of oogenesis is rich with important developmental and morphogenetic processes.
Isolated mid-to-late stage follicles (S10B-S14) can be used for a variety of purposes, including molecular analyses. For example, mRNA from staged follicles can be isolated for RT-PCR, microarray, or RNA-seq analyses. This allows one to look at gene expression within a short developmental window, with only a few cell types present, and determine how gene expression is changed by either pharmacologic or genetic perturbations. Stage isolation can also be used to look at proteins by western blotting. Such analysis is important because it allows one to quantify the level of protein expression in wild-type versus mutants at specific stages. While one could use immunofluorescent analyses to achieve similar results, quantification of fluorescence is less robust due to the strict requirements that all of the pixels be within the linear range of detection6. Additionally, western blot analysis may provide other information, such as if the protein is posttranslationally modified or is expressed from a specific splice isoform. Isolated stages can also be used for further protein purification, including subcellular fractionation or coimmunoprecipitation.
Stage-specific follicle isolation can also be used for in vitro development assays7 and live-imaging8. Isolated S10B-S13 follicles will continue to develop to S14 in simple culture media (see below). It is important to note that S10A follicles will not progress through nurse cell dumping using the culture conditions discussed in this manuscript. We have used S10B in vitro development assays to define the role of prostaglandins, both pharmacologically and genetically, in regulating actin remodeling by using nurse cell dumping and development as read-outs7,9. Similarly, the later stages of development can also be isolated to determine the effects of pharmacologic treatments or genetic manipulations on particular processes such as centripetal cell migration, dorsal appendage migration/formation10, and nurse cell death. Such assays can be used to perform dominant interaction screens or assays; for example, while heterozygosity for mutations in pxt or fascin alone have no effect on S10B in vitro development, follicles from double heterozygotes exhibit nurse cell dumping defects and a block in development9.
Additionally, because S10B-13 can develop in culture, all of the processes that occur during this time can be observed by live-imaging. Such imaging can be performed simply using transmitted light (if one is only interested in gross changes in morphology) or with confocal microscopy using transgenic flies expressing fluorescent probes or follicles stained with live imaging dyes. Live imaging is being used to substantially advance our understanding of developmental processes. Indeed, live imaging of late stage follicles has expanded the knowledge of dorsal appendage migration, an example of tubulogenesis10. We expect that live imaging of additional late stage processes, including actin dynamics during nurse cell dumping, will provide novel insights into these developmental events. It is important to note that while S10A and early stages of follicle development will not continue to develop into a S14 in culture, live-imaging of events occurring during those stages of development is possible using alternative culture conditions11-14 (see Discussion for more information).
Here we provide detailed protocols for isolating late stage follicles for either in vitro development and live-imaging, or molecular analyses (mRNA and protein isolation).

<table border="0" cellpadding="0" cellspacing="0" width="1098">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:253px;">Active Dry Yeast</td>
			<td style="width:195px;">Genesee Scientific</td>
			<td style="width:227px;">62-103</td>
			<td style="width:424px;">Any source of Active Dry Yeast is fine</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Grace&#8217;s Insect Media</td>
			<td>Lonza</td>
			<td>04-457F</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050H</td>
			<td>Any Heat Inactivated FBS should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10x Pen/Strep</td>
			<td>Gibco/Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pin Vises and Needles</td>
			<td>Ted Pella, Inc.</td>
			<td>13561-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spot Plate, Nine Well</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">#5 Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24 multi-well plates</td>
			<td>Becton Dickinson</td>
			<td>35 3226</td>
			<td>Any 24-well tissue culture dish should work</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coverslip Bottom Dishes (35mm)</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Coverslip thickness will depend on the microscope/objective being used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes</td>
			<td>Corning</td>
			<td colspan="2">7095B-5x (for transferring follicles)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glass pipettes - long</td>
			<td>Corning</td>
			<td colspan="2">7095B-9 (for producing pulled pipettes)</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Sample pestle (1.5 &#956;l; RNase/DNase free)</td>
			<td>Research Products International</td>
			<td>199228</td>
			<td>Any plastic pestle that fits 1.5 &#956;l microfuge tubes can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td></td>
		</tr>
	</tbody>
</table>

the utility of stage-specific mid-to-late drosophila follicle isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

When isolating specific stages of Drosophila follicle development it is essential to be able to accurately distinguish the different morphological stages. This is somewhat challenging for S10A and S10B, as the nurse cells and the oocyte each take up half the length of the follicle at these stages (Figure&#160;1B compared to 1C). However, S10A follicles are shorter in length than S10B follicles, as the S10B follicles are fully elongated and thus equal in length to a S14 follicle ...

Watch this Scientific Journal Video about The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation at JoVE.com

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

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