Name: visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers
Uploaded: 2019-06-04T12:30:00
Description: Watch this Scientific Journal Video about Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers at JoVE.com

We thank Salvatore A.E. Marras (Public Health Research Institute Center, Rutgers University) for the synthesis, labeling and purification of molecular beacons, and Daniel St Johnston (The Gurdon Institute, University of Cambridge) for the oskar-MS2/MCP-GFP transgenic fly stock. This work was supported by a National Science Foundation CAREER Award 1149738 and a Professional Staff Congress-CUNY Award to DPB.

1. Design of MBs for Live Cell Imaging
<ol>
	<li>Fold the target RNA sequence to predict the mRNA target&#8217;s secondary structure using the &#8220;RNA form&#8221; from the mfold server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form).
	<ol>
		<li>Paste/upload the target sequence in FASTA format, select 5 or 10% sub-optimality (structures with a free energy of folding within 5 or 10% of the MFE value, respectively), and adjust the maximum number of computed foldings accordingly (e.g...

<ol>
	<li>Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+RNA+distribution+and+dynamics+in+living+cells.">Imaging intracellular RNA distribution and dynamics in living cells.</a> Nature Methods. 6 (5), 331-338 (2009).</li><li>Bao, G., Rhee, W. J., Tsourkas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+for+live-cell+RNA+detection.">Fluorescent probes for live-cell RNA detection.</a> Annual Reviews of Biomedical Engineering. 11, 25-47 (2009).</li><li>Mannack, L. V., Eising, S., Rentmeister, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+techniques+for+visualizing+RNA+in+cells.">Current techniques for visualizing RNA in cells.</a> F1000Research. 5, (2016).</li><li>Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A., Singer, R. 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F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS2+coat+proteins+bound+to+yeast+mRNAs+block+5'+to+3'+degradation+and+trap+mRNA+decay+products:+implications+for+the+localization+of+mRNAs+by+MS2-MCP+system.">MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system.</a> RNA. 21 (8), 1393-1395 (2015).</li><li>Bratu, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacons:+Fluorescent+probes+for+detection+of+endogenous+mRNAs+in+living+cells.">Molecular beacons: Fluorescent probes for detection of endogenous mRNAs in living cells.</a> Methods in Molecular Biology. 319, 1-14 (2006).</li><li>Bratu, D. P., Cha, B. J., Mhlanga, M. M., Kramer, F. 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Live visualization of endogenous mRNA trafficking in Drosophila egg chambers relies on the use of specific, efficient, and nuclease-resistant MBs, which can now be easily designed with PinMol software. MBs are specific probes designed to detect unique sequences within a target mRNA (preferably regions free of secondary structure), making possible highly resolved detection of a transcript. The only limitation when adopting this technique/protocol for other tissues/cell types is the efficiency of MB deliv...

Cell biology studies that visualize dynamic events with spatial and temporal resolution have been made possible by the development of fluorescence-based live cell imaging techniques. Presently, in vivo mRNA visualization is achieved via technologies that are based on RNA aptamer-protein interactions, RNA aptamer-induced fluorescence of organic dyes and nucleic acid probe annealing1,2,3. They all offer high specificity, sensitivity and signal-to-background ratio. However, RNA aptamer-centered approaches require extensive genetic manipulation, where a transgene is engineered to express an RNA with artificial structural motifs that are required for protein or organic dye binding. For example, the MS2/MCP system requires the co-expression of a transgene expressing an RNA construct containing multiple tandem repeats of the binding sequence for the bacteriophage MS2 coat protein (MCP), and another transgene encoding a fluorescent protein fused to MCP4,5. The addition of such secondary structural motifs to the RNA, along with a bulky fluorescently tagged protein, has raised concerns that native RNA processes may be affected6. A technology that addresses this concern and offers additional unique advantages is the nucleic acid-based approach, molecular beacons (MBs). MBs allow for the multiplex detection of endogenous mRNAs, discrimination of single nucleotide variations, and fast kinetics of hybridization with target mRNA7,8. MBs are oligonucleotide probes that remain in a quenched hairpin fold prior to undergoing a fluorogenic conformational change once they hybridize to their targets (Figure 1C)9. Several groups have had success in using MBs to detect both non-coding RNAs (microRNAs and lncRNAs)10,11,12,13, RNA retroviruses14 and dynamic DNA-protein interactions15. They have been successfully employed for imaging in various organisms and tissues, such as zebrafish embryos16, neurons13, tumor tissue17, differentiating cardiomyocytes18, and Salmonella19.
Here we describe the design, delivery and detection approach for endogenous mRNAs in living D. melanogaster egg chambers coupled with a microscopy set-up that is fast enough to capture the dynamic range of active molecular transport. The D. melanogaster egg chamber has served as an ideal multicellular model system for a wide range of developmental studies, from early germline stem cell division and maternal gene expression to the generation of segmental body plan20,21. Egg chambers are easily isolated, large and translucent, and able to withstand hours of ex vivo analysis, making them highly amenable to imaging experiments. Much work has focused on the asymmetric localization of maternal transcripts to discrete subcellular regions prior to being actively translated. In particular, oskar mRNA localization and its subsequent translation at the oocyte&#39;s posterior pole must occur in a tightly regulated manner to avoid a lethal bicaudal embryo phenotype22. oskar mRNA is transcribed in the 15 germline cells, called nurse cells, and actively transported through cytoplasmic bridges, called ring canals, into the oocyte, the germline cell that becomes the mature egg and is ultimately fertilized (Figure 1A). The considerable amount of information already available regarding the dynamic recruitment and exchange of protein factors to and from oskar mRNP, along with its long-range intracellular travel, make oskar a preferred candidate to study the many processes of the mRNA life cycle. MBs have been instrumental in revealing details about the process of mRNA localization and deciphering the regulation and function of protein factors that control mRNA transport during Drosophila oogenesis. In particular, by microinjecting MBs into nurse cells and performing live cell imaging experiments, the tracking of endogenous mRNAs is possible8,23.
The roadmap presented here offers the steps of a complete process, from carrying out a live cell imaging experiment using MBs, acquiring imaging data, to performing data analysis to track endogenous mRNA in its native cellular environment. The steps can be modified and further optimized to meet the needs of researchers working with other tissues/cell types within their own lab setting.

<table>
	<tbody>
		<tr>
			<td>Spectrofluorometer</td>
			<td>Fluoromax-4 Horiba-Jobin Yvon</td>
			<td>n/a</td>
			<td>Photon counting spectrofluorometer</td>
		</tr>
		<tr>
			<td>Quartz cuvette</td>
			<td>Fireflysci (former Precision Cells Inc.)</td>
			<td>701MFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 tweezer</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td>Thin tweezers are very important to separate out the individual egg chambers</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip No.1 22 x 40mm</td>
			<td>VWR</td>
			<td>48393-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6 Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 fruit fly anesthesia pad</td>
			<td>Genesee Scienific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL pH7.5</td>
			<td>Sigma-Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope</td>
			<td>Leica DMI-4000B inverted microscope equipped with Yokogawa CSU 10 spinning disc Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu C9100-13 ImagEM EMCCD camera</td>
			<td>Hamamatsu</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PatchMan NP 2 Micromanipulator</td>
			<td>Eppendorf Inc.</td>
			<td>920000037</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector</td>
			<td>Eppendorf Inc.</td>
			<td>920010504</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle: Femtotips II</td>
			<td>Eppendorf Inc.</td>
			<td>930000043</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading tip: 20ul Microloader</td>
			<td>Eppendorf Inc.</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover glasses no. 1 or 1.5, 22x40mm</td>
			<td>VWR</td>
			<td>48393-026; 48393-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry yeast</td>
			<td>Any grocery store</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer, &#62; 20 GB RAM</td>
			<td></td>
			<td></td>
			<td>Although processing can be carried out on most computers, higher capabilities will increase the speed of the processing</td>
		</tr>
	</tbody>
</table>

visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Using PinMol, several MBs can be designed for one mRNA target (Figure 1B-C). After synthesis and purification, the selected MBs are characterized and compared using in vitro analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58545/58545fig1.jpg" /> 
Figure 1: Technique and tissue description for live cell imaging of endogenous mRNAs. ...

Watch this Scientific Journal Video about Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers at JoVE.com

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

This method can help answer key questions in the RNA biology field about the mechanism and function of polarized RNA localization. The main advantage of this technique is that it allows for the real time visualization of endogenous RNA trafficking in live tissues. The implications of this technique extend toward the therapy of RNA-related diseases because it has a potential to uncover new therapeutic targets.Though this method can provide insight into RNA processes in the fruit fly egg chamber, it can also be applied to other systems such as in zebrafish or culture cells. Generally, individuals new to this method will struggle because of the challenges associated with the design and delivery of the molecular beacon props. For molecular beacon microinjection, select the 40X oil objective and mount a coverslip with the dissected egg chamber onto the microscope stage.Locate an egg chamber at the mid to late developmental stage that is properly oriented for microinjection. Then set up a microinjector with the appropriate injection and compensation pressures. Using the micro manipulator joystick, gently lower a needle loaded with one microliter of molecular beacon solution into the oil drop.Bring the tip into focus toward the periphery of the field of view. Perform a clean function to remove the air from the needle tip and to ensure that there is flow from the needle and bring the needle to the home position. Focus on the egg chamber to be microinjected and bring the needle back into focus near the edge of the egg chamber.Perform a fine adjustment of the z-position of the objective such that the membrane separating the follicle cells from the nurse cells is in focus. Insert the needle into a nurse cell for injection of the molecular beacons over a period of two to five seconds. To ensure reproducible microinjection success, focus carefully on the membrane separating the follicle cells and the nurse cells that will be injected while bringing the tip of the needle into focus with the micro manipulator.When all of the molecular beacons have been injected, gently remove the needle and retract it to the home position. Change the objective to the desired magnification for image acquisition. Then focus on the egg chamber under the new objective and begin the image acquisition.For spot detection of the molecular beacons, open the images in Image J.Use the convert to ICY tool to convert the images back to ICY. A scale bar will be automatically overlaid onto the stack. Edit the scale bar via the inspector window as necessary.Then inactivate the eye icon for the scale bar under the layer tab to remove the scale bar from the original stack. Select detection and tracking, detection, and spot detector, and confirm current sequence input detection and channel zero are selected for the input and preprocessing parameters respectively. For detector, select detect bright spot over dark background using force use of 2D wavelengths for 3D only if there are not enough z-slices in the stacks to perform the analysis.Select scales and sensitivity for each scale adding more scales for larger spots and confirm that region of interest from sequence is set for region of interest. For filtering, confirm that no filtering has been set and select the appropriate file format under output. If the spot detector results are to be used for tracking analysis, select export to swimming pool.For colocalization analysis, repeat the spot detection for the other channel. To track the molecular beacon spots, select detection and tracking, tracking, spot tracking, and run the spot detector using the same parameters as for the spot detection. Click estimate parameters and select the desired target motion in the parameters estimation popup window.Then click run tracking. To visualize the tracks, select detection and tracking, tracking, and track manager. For color track processor, select enable and select a color for the tracks.From add track processor, select track processor time clip. In the check clipper window, enter the number of detections to be shown before and after the current time point. Save the track information as an XML track file and use the camera icon to obtain a screenshot of the results.To project the stack along the z-direction, use the search bar to find the plugin and select maximum projection from the dropdown menu. In the inspector window, select the sequence tab, canvas, and rotation to adjust the image to the desired orientation and obtain a screenshot of the rotated image. Then select region of interest, 2D region of interest, choose region of interest shape, and select the region of interest on the image for cropping.Install the timestamp overlay plugin and add a timestamp following the instructions in the popup window for directions on how to place and format the timestamp. To change or add the time interval, click edit in the sequence properties window. Activate the eye icon to add back the scale bar under the layer tab in the inspector window.Then obtain a screenshot of the timestamped results and save the image in both TIFF and AVI formats. Using this method as demonstrated, it is possible to visualize the transport and localization patterns of endogenous mRNAs via molecular beacons at various stages of oogenesis and in particular at and after mid oogenesis. When individually injected into the same stage egg chambers, different oskar specific molecular beacons present the same pattern of localization.Molecular beacons injected into the cytoplasm of a nurse cell will freely diffuse into adjacent nurse cells as well as into the oocyte. Thus, the beacons are able to hybridize with their targets and generate fluorescence signals at other sites than the microinjection site. In oskar GFP transgenic egg chambers at mid oogenesis, analysis of the acquisition data shows extensive colocalization for the fluorescence signal of genetically engineered GFP tagged oskar mRNA with the fluorescence signal detected using molecular beacons.Moreover, 5D stacks can be further analyzed to determine oskar mRNA trajectories for long distance transport in both nurse cell and oocyte cytoplasm. After its development, this technique paved the way for researchers in the field of RNA biology to explore endogenous maternal mRNA transport and localization in the Drosophila egg chamber.

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