Our work on RNA localization is supported by a grant from the NIH (R01GM071049) to KLM.

Part 1: Transcription of Fluorescently Labeled mRNA.
<ol>
 <li>Linearize plasmid DNA containing the RNA localization element or other relevant sequence and resuspend at 1 &mu;g/&mu;l with DEPC-treated H2O. The DNA template must have upstream promoter sites for transcription by T7, SP6, or T3 RNA polymerase.</li>
 <li>Add the following reagents to a sterile 1.5mL tube:
 <table>
 <tbody>
 <tr><td>a. 10X Tx buffer (see M&amp;M)</td><td>2 &mu;l</td></tr>
 <tr><td>b. 20X cap/NTP mix (see M&amp;M)</td><td>1 &mu;l</td></tr>
 <tr><td>c. 1 mM Alexa Fluor 546-14-UTP (Invitrogen)</td><td>1 &mu;l</td></tr>
 <tr><td>d. UTP, [&alpha;-32P](1 &mu;Ci/&mu;l) (Perkin Elmer)</td><td>1 &mu;l</td></tr>
 <tr><td>e. DEPC-H2O</td><td>11 &mu;l</td></tr>
 <tr><td>f. 0.2 M DTT</td><td>1 &mu;l</td></tr>
 <tr><td>g. RNasin (Promega)</td><td>1 &mu;l</td></tr>
 <tr><td>h. linear template DNA</td><td>1 &mu;l</td></tr>
 <tr><td>i. RNA Polymerase (Promega)</td><td>1 &mu;l</td></tr>
 </tbody>
 </table>
 </li> 
 <li>Mix reagents gently and centrifuge briefly (10 sec. in a microcentrifuge).</li>
 <li>Incubate for 2-4 hours at 37&deg; C, covering the tube with aluminum foil to prevent photobleaching of fluorophore.</li>
 <li>Add 1 &mu;l 1 mg/ml RNase-free DNase (Promega) to degrade template DNA.</li>
 <li>Incubate 15 minutes at 37&deg; C.</li>
 <li>Add 79 &mu;l 20 mM EDTA (pH 8.0) to stop the reaction.</li>
 <li>Remove 1 &mu;l to a separate tube, labeled as "input" and save.</li>
 <li>Spin reaction through a 1 ml G50 column (see Materials &amp; Methods), which will include unincorporated nucleotides while excluding full length mRNA.</li>
 <li>Concentrate the RNA by ethanol precipitation:
 <ol class="lalpha">
 <li>Add 250 &mu;l 100% ethanol, 10 &mu;l 7M CH3COONH4, 1 &mu;l carrier RNA (yeast tRNA, 10 &mu;g/&mu;l) or 1 &mu;l glycogen (20 mg/ml).</li>
 <li>Mix well and freeze until solid at -80&deg; C for &gt;30 minutes or on dry ice for 15 minutes. </li>
 <li>Spin at maximum speed in microcentrifuge for 15 minutes, decant supernatant.</li>
 <li>Wash with 150 &mu;l of 75% ethanol.</li>
 <li>Spin for 3 minutes at maximum speed in microcentrifuge, decant supernatant.</li>
 <li>Dry the pellet at 37&deg; C for 3 minutes, ensuring that all ethanol has evaporated.</li>
 </ol>
 </li>
 <li>Resuspend in 11 &mu;l DEPC-H2O and remove 1 &mu;l to a separate tube, labeled as "incorporated".</li>
 <li>Determine the percent incorporation and dilute the RNA to 50 nM (see Materials &amp; Methods for calculation). </li>
 <li>The RNA should be frozen in 5 &mu;l aliquots, for single use to avoid freeze / thaw cycles and can be stored for several weeks at -80&deg; C.</li>
</ol>
Part 2: Preparing for Microinjection.
<ol>
 <li>RNA Preparation: Thaw an aliquot of RNA (50 nM), and denature the RNA at 70&deg; C for 3-5 minutes. Spin for 10 minutes in room temperature microcentrifuge at maximum speed to remove any particulates, and keep on ice.</li>
 <li>Oocyte Preparation:
 <ol class="lalpha">
 <li>Surgically remove ovary from Xenopus laevis females (Nasco). </li>
 <li>Trim pieces of Xenopus laevis ovary into a 50 ml conical tube containing 25 ml of Collagenase solution (see Materials &amp; Methods). </li>
 <li>Shake gently at 18&deg; C for 15 minutes, or until oocytes are visibly separated from ovary. Due to batch to batch variation, collagenase treatment time may vary and should be monitored carefully through visual inspection to ensure defoliculation. </li>
 <li>Allow oocytes to settle in tube, remove solution and wash the oocytes with MBSH buffer (see Materials &amp; Methods). </li>
 <li>Repeat wash twice more for a total of three washes. This oocyte isolation protocol is similar to that detailed in Cohen et al., 2009. </li>
 <li>Manually sort stage III/IV oocytes (Dumont, 1972) in MBSH buffer under a standard dissecting microscope. Stage III oocytes are completely opaque but white, while stage IV oocytes are slightly larger and speckled with pigment. Oocytes that are slightly transparent are too young and oocytes that fully pigmented or exhibit polarized pigment distribution are too old.</li>
 </ol>
 </li>
 <li>Needle Preparation: Pull and bevel needles to an outer diameter of ~ 0.05 mm. (We use Drummond Scientific 3.5 inch capillaries (order # 3-000-203-G/X), a Sutter Instrument Co. micropipette puller and a WPI Inc. beveller.)</li>
</ol>
Part 3: Microinjection
<ol>
 <li>Calibrate needle with DEPC-H2O to deliver 2 nl per injection. We front load our needle using a gas-driven microinjector (see Materials &amp; Methods), and calibrate drop size using a micrometer. </li>
 <li>Load RNA into needle.</li>
 <li>Place sorted oocytes into an injection dish containing MBSH buffer. We microinject on a dish with a layer of black foam rubber. The pale oocytes stand out well on a white background.</li>
 <li>Carefully inject each oocyte with 2 nl of RNA at 50 nM.</li>
 <li>Expel RNA, rinse needle with DEPC-H2O, and load next RNA for injection.</li>
</ol>
Part 4: Oocyte Culture
<ol>
 <li>Place oocytes in a well of a sterile 24 well plate (Sigma Aldrich). We culture up to a thousand oocytes per well. </li>
 <li>Remove buffer and replace with 400 &mu;l Oocyte Culture Medium (OCM; see Materials &amp; Methods) per well. Place culture plate inside an air-tight plastic container with wet paper towel to maintain moist environment during culture.</li>
 <li>Incubate oocytes at 18&deg; C for time points ranging between 8 and 48 hours.</li>
</ol>
Part 5: Fixation, Dehydration and Storage of Oocytes
<ol>
 <li>Sort out any dead oocytes and place surviving oocytes in glass vials. We routinely observe &gt;90% survival. </li>
 <li>Place oocytes in MEMFA fixative (see Materials &amp; Methods) and rock for 20 minutes. Protect the oocytes from light by covering with aluminum foil.</li>
 <li>Wash oocytes by removing fixative and replacing with equal volume of MBSH buffer. Repeat this wash once more for a total of two washes.</li>
 <li>Wash oocytes into anhydrous methanol:
 <ol class="lalpha">
 <li>Remove half of the volume, replace with methanol.</li>
 <li>Repeat step "a" twice.</li>
 <li>Remove all of the solution, replace with methanol.</li>
 <li>Repeat methanol wash.</li>
 </ol>
 </li>
 <li>Oocytes can be stored at -20&deg; C until ready to image.</li>
</ol>
Part 6: Imaging RNA Localization by Confocal Microscopy
<ol>
 <li>Before imaging, add Murray s Clearing Medium (2:1 benzyl benzoate-benzyl alcohol) to a glass bottom FluoroDish (WPI Inc.) to cover the imaging surface. </li>
 <li>Carefully transfer the oocytes from methanol to FluoroDish, minimizing the volume of methanol transferred.</li>
 <li>Image on an inverted confocal microscope (We have successfully used a Zeiss LSM510 and a Leica TCS SP2).</li>
</ol>
<img src="/files/ftp_upload/1704/1704fig1.jpg" alt="Figure 1" /> Figure 1: Visualization of Subcellular RNA Localization by Microinjection of Fluorescently Labelled Transcripts.&nbsp; A.&nbsp; Following microinjection, RNA labeled with Alexa Fluor 546 is initially restricted to the nucleus. B. After eight hours of culture, a fluorescently labeled control RNA can be seen uniformly distributed in the cytoplasm of the oocyte. C. However, fluorescently labeled RNA containing sequences that recruit the transport machinery can be seen in the process of subcellular localization to the vegetal pole of the oocyte (towards bottom). Scale bar = 50 &mu;m.

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

Experiments using laboratory animals were performed in accordance with the guidelines and regulations set forth by Brown University.

Here we have presented a protocol for visualizing mRNA localization in Xenopus oocytes. This method, using fluorescently labeled RNA transcripts has higher signal to noise ratio than previously obtained with digoxigenin-labeled transcripts and is simpler and faster than in-situ based approaches (Mowry and Melton, 1992, Gautreau et al., 1997). Using this method, we can engineer RNA sequence mutations and rapidly test for in vivo function. Further, by using additional Alexa-UTP fluorophores, multiple RNA ...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X Tx buffer
<ul>
<li class="none">60 mM MgCl2</li>
<li class="none">400 mM Tris-HCl (pH 7.5)</li>
<li class="none">20 mM spermidine-HCl</li>
</ul>
20x cap/NTP mix
<ul>
<li class="none">10 mM CTP</li>
<li class="none">10 mM ATP</li>
<li class="none">9 mM UTP</li>
<li class="none">2 mM GTP</li>
<li class="none">20 mM G(ppp)G Cap Analog (New England Biolabs)</li>
</ul>
G-50 column
<ul>
<li class="none">Hydrate 5 g Sephadex G-50 beads (Sigma Aldrich) in 100 ml deionized H2O. DEPC-treat for 30 min. and autoclave. Store incomplete stock at room temperature. Before use, add the following RNase-free solutions:</li>
<li class="none">0.5 ml 0.2 M EDTA</li>
<li class="none">1 ml 1 M Tris pH 8.0</li>
<li class="none">0.5 ml 20% SDS</li>
<li class="none">Store complete G-50 solution at 4&deg; C.</li>
<li class="none">Remove and discard the plunger from a 3 ml syringe (BD Biosciences) and place the barrel of the syringe into a 15 ml conical tube (Corning). Plug the syringe with a small amount of glass wool (a plug about half the size of a penny).</li>
<li class="none">Swirl complete G-50 solution to resuspend beads.</li>
<li class="none">Add 2 ml G-50 solution to the empty column.</li>
<li class="none">Spin for 1 minute at 1,000 x g in benchtop centrifuge.</li>
<li class="none">Add 200 &mu;l DEPC-treated deionized H2O to each column. Spin.</li>
<li class="none">Repeat wash twice more for a total of three washes.</li>
<li class="none">Remove syringe barrel to a fresh 15 ml conical tube.</li>
</ul>
Collagenase solution
<ul>
<li class="none">75 mg collagenase from Clostridium histolyticum (Sigma Aldrich)</li>
<li class="none">25 ml 0.1 M KPO3+ (pH 7.4)</li>
</ul>
MBSH buffer
<ul>
<li class="none">88 mM NaCl</li>
<li class="none">1 mM KCl</li>
<li class="none">2.4 mM NaHCO3</li>
<li class="none">0.82 mM MgSO4 X 7H2O</li>
<li class="none">0.33 mM Ca(NO3)2 X 4H2O</li>
<li class="none">0.41 mM CaCl2 X 6H2O</li>
<li class="none">10 mM HEPES (pH 7.6)</li>
</ul>
Oocyte Culture Medium
<ul>
<li class="none">50% L15 medium</li>
<li class="none">15 mM HEPES (pH 7.6)</li>
<li class="none">1 mg/ml insulin</li>
<li class="none">100 mg/ml gentamicin</li>
<li class="none">50 U/ml nystatin</li>
<li class="none">50 U/ml penicillin</li>
<li class="none">50 mg/ml streptomycin</li>
</ul>
MEMFA solution
<ul>
<li class="none">0.1 M MOPS (pH 7.4)</li>
<li class="none">2 mM EGTA</li>
<li class="none">1 mM MgSO4</li>
<li class="none">3.7% formaldehyde</li>
</ul>
Computing RNA yield
<ul>
<li class="none">Determine CPM in "input" and "incorporated" samples using a standard scintillation counter.</li>
<li class="none">incorporation = ("incorporated") / (10 x "input")</li>
<li class="none">Typical incorporation values range between ~0.03 and 0.10.</li>
<li class="none">Maximum theoretical yields for different polymerases: 
T7, T3, SP6 - 2.64 &mu;g</li>
<li class="none">Reaction yield in &mu;g = (maximum yield of polymerase used) X (incorporation)</li>
<li class="none">Dilute RNA to 50 nM = (&mu;g RNA) / 320 / (length of RNA in bases) / (5X10-8)</li>
<li class="none">The reaction usually yields ~50-100 &mu;l of RNA at 50 nM.</li>
</ul>

visualizing rna localization in xenopus oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

11.5K ビュー.  Brown University. の可視化 in vivoで RNAのトランスポートは、蛍光標識RNA転写物のマイクロインジェクションによってに行われますアフリカツメガエル卵母細胞、共焦点顕微鏡に続く。

ビデオ: Visualizing RNA Localization in Xenopus Oocytes

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Genetics

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Developmental Biology

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

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.

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.

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

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.