Name: in situ detection of ribonucleoprotein complex assembly in the c. elegans germline using proximity ligation assay
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Description: Watch this Scientific Journal Video about In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay at JoVE.com

Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center funded by the NIH (P40OD010440). Confocal microscopy was performed in the University of Montana BioSpectroscopy Core Research Laboratory operated with support from NIH Awards P20GM103546 and S10OD021806. This work was supported in part by the NIH grant GM109053 to E.V., American Heart Association Fellowship 18PRE34070028 to X.W., and Montana Academy of Sciences award to X.W. The funders were not involved in study design or writing the report. We thank M. Ellenbecker for discussion.

NOTE: This protocol uses C. elegans strains in which potential interacting partners are both tagged. It is strongly recommended that a negative control strain be used, in which one tagged protein is not expected to interact with another tagged candidate interaction partner. Here, GFP alone was used as a negative control to assess background, as DLC-1 is not expected to interact with GFP in the worm. GFP-tagged OMA-1 was used as the experimental strain, as preliminary data suggest an interaction with DLC-1. Nemat...

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When studying PPIs in the C. elegans germline, the higher resolution offered by PLA compared to co-immunostaining allows visualization and quantification of locations where interactions occur in the germline. It was previously reported that DLC-1 directly interacts with OMA-1 using an in vitro GST pulldown assay26; however, this interaction was not recovered by an in vivo pulldown. The fluorescent co-immunostaining of 3xFLAG::DLC-1; OMA-1::GFP germlines shows an overlap in the ex...

Over 80% of proteins are estimated to have interactions with other molecules1, which emphasizes how important PPIs are to the execution of specific biological functions in the cell2. Some proteins function as hubs facilitating assembly of larger complexes that are necessary for cell survival1. These hubs mediate multiple PPIs and help organize proteins into a network that facilitates specific functions in a cell3. Formation of protein complexes is also affected by biological context, such as the presence or absence of specific interacting partners4, cell signaling events, and developmental stage of a cell.
C. elegans is commonly used as a model organism for a variety of studies, including development. The simple anatomy of this animal is comprised of several organs, including the gonad, gut, and transparent cuticle, which facilitates the analysis of worm development. The germline residing in the gonad is a great tool to study how germline stem cells mature into gametes5 that develop into embryos and eventually the next generation of progeny. The distal tip region of the germline contains a pool of self-renewing stem cells (Figure 1). As stem cells leave the niche, they progress into the meiotic pachytene and eventually develop into oocytes in the young adult stage (Figure 1). This program of development in the germline is tightly regulated through different mechanisms, including a post-transcriptional regulatory network facilitated by RNA-binding proteins (RBPs)6. PPIs are important for this regulatory activity, as RBPs associate with other cofactors to exert their functions.
There are several approaches that can be used to probe for PPIs in the worm, but each has unique limitations. In vivo immunoprecipitation (IP) can be used to isolate protein-protein complexes from whole worm extracts; however, this approach does not indicate where the PPI occurs in the worm. In addition, protein complexes that are transient and only form during a specific stage of development or in a limited number of cells can be difficult to recover by co-immunoprecipitation. Finally, IP experiments need to address the concerns of protein complex reassortment after lysis and non-specific retention of proteins on the affinity matrix.
Alternative approaches for in situ detection of PPIs are co-immunostaining, F&#246;rster resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC). Co-immunostaining relies on simultaneous detection of two proteins of interest in fixed worm tissue and measurement of the extent of signal colocalization. Use of super-resolution microscopy, which offers greater detail than standard microscopy7, helps to more stringently test protein colocalization beyond the diffraction-limited barrier of 200-300 nm8. However, co-immunostaining using both conventional and super-resolution microscopy works best for proteins with well-defined localization patterns. By contrast, it becomes much less informative for diffusely distributed interacting partners. Measuring for co-localization of signals based on overlap does not provide accurate information about whether the proteins are in complex with each other9,10.
Furthermore, co-immunoprecipitation and co-immunostaining of protein-protein complexes are not quantitative, making it challenging to determine if such interactions are significant. FRET and BiFC are both fluorescent-based techniques. FRET relies on tagging proteins of interest with fluorescent proteins (FPs) that have spectral overlap at which energy from one FP (donor) is transferred to another FP (acceptor)11. This nonradiative transfer of energy results in fluorescence of the acceptor FP that can be detected at its respective wavelength of emission. BiFC is based on reconstitution of a fluorescent protein in vivo. It entails splitting GFP into two complementary fragments, such as helices 1-10 and helix 1112, which are then fused to two proteins of interest. If these two proteins interact, the complementary fragments of GFP become close enough in proximity to fold and assemble, reconstituting the GFP fluorophore. Reconstituted GFP is then directly observed as fluorescence and indicates where a PPI has occurred.
As such, both FRET and BiFC depend on large fluorescent tags that can disrupt the function of the tagged protein. In addition, FRET and BiFC require abundant and comparable expression of the tagged proteins to obtain accurate data. FRET may not be suitable for experiments where one partner is in excess of the other, which can lead to high background13. Overexpression in BiFC experiments should also be avoided, as this can induce nonspecific assembly14 that results in increased background. Both techniques require optimization of expression and imaging conditions of the tagged proteins, which may prolong the time required to complete experiments.
The proximity ligation assay (PLA) is an alternative approach that can address the limitations of the techniques mentioned above. PLA takes advantage of primary antibodies that recognize the proteins of interest (or their tags). These primary antibodies are then bound by secondary antibodies containing oligonucleotide probes that can hybridize with one another when within a 40 nm (or shorter) distance15. The resulting hybridized DNA is amplified through a PCR reaction, which is detected by probes that complement the DNA. This results in foci that are visualized by a microscope. This technology can detect PPIs in situ in complex tissues (i.e., the worm gonad), which is organized as an assembly line containing cells at various stages of development and differentiation. With PLA, PPIs can be directly visualized in a fixed worm gonad, which is advantageous for investigating whether PPIs occur during a specific stage of development. PLA offers greater resolution of PPIs as opposed to co-localization-based assays, which is ideal for making precise measurements. If used, super-resolution microscopy has the potential to provide finer detail about the location of PLA foci within a cell. Another advantage is that the foci resulting from PLA reactions can be counted by an ImageJ-based analysis workflow, making this technique quantitative.
The LC8 family of dynein light chains was first described as a subunit of the dynein motor complex16 and hypothesized to serve as a cargo adapter. Since its initial discovery, LC8 has been found in multiple protein complexes in addition to the dynein motor complex17,18,19,20. Scanning for protein sequences that contain the LC8 interaction motif19 suggests that LC8 may have many interactions with a wide array of different proteins17,18,19,20,21,22. As a result, LC8 family proteins are now considered hubs that help promote the assembly of larger protein complexes19,22, such as assemblies of intrinsically disordered proteins21.
One C. elegans LC8-family protein, dynein light chain-1 (DLC-1), is widely expressed across many tissues and not enriched in specific subcellular structures23,24. Consequently, identification of biologically relevant in vivo partners of DLC-1 in C. elegans is challenging for a number of reasons: 1) co-immunoprecipitation does not indicate the tissue source where the interaction occurs; 2) limited expression of particular partners or transient interactions may hinder the ability to detect an interaction by co-immunoprecipitation; and 3) diffuse distribution of DLC-1 leads to non-specific overlap with potential partner proteins by co-immunostaining. Based on these challenges, PLA is an ideal approach for testing in vivo interactions with DLC-1.
It has been previously reported that DLC-1 directly interacts with and serves as a cofactor for the RNA-binding proteins (RBPs) FBF-223 and GLD-125. Our work supports the model of DLC-1 serving as a hub protein and suggests that DLC-1 facilitates an interaction network that spans beyond dynein19,22. Using a GST pulldown assay, a new DLC-1-interacting RBP named OMA-1 has been identified26. OMA-1 is important for oocyte growth and meiotic maturation27 and functions in conjunction with a number of translational repressors and activators28. While FBF-2 and GLD-1 are expressed in the stem cells and meiotic pachytene regions, respectively, OMA-1 is diffusely expressed in the germline from the meiotic pachytene through the oocytes27 (Figure 1). This suggests that DLC-1 forms complexes with RBPs in different regions of the gonad. It has also been found that the direct interaction between DLC-1 and OMA-1 observed in vitro is not recovered by an in vivo IP. The PLA has been successfully used as an alternate approach to further study this interaction in the C. elegans germline, and results suggest that PLA can be used to probe many other PPIs in the worm.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% paraformaldehyde solution</td>
			<td>Electron Microscopy services</td>
			<td>15710</td>
			<td>used to make 4% working solution</td>
		</tr>
		<tr>
			<td>1M KH2PO4</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td>Prepare a 1M working stock</td>
		</tr>
		<tr>
			<td>1x M9</td>
			<td>various</td>
			<td>various</td>
			<td>prepared as 10x stock used at 1x; see wormbook.org for protocol</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>various</td>
			<td>various</td>
			<td>see wormbook.org for protocol</td>
		</tr>
		<tr>
			<td>26.5 Gauge Needle</td>
			<td>Exel International</td>
			<td>26402</td>
			<td>Needles used for dissection</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Lampire</td>
			<td>7500802</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Thermo Scientific</td>
			<td>05-529C</td>
			<td>50ml Oak ridge centrifuge tube used for synchronization</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>880</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>PolyLab</td>
			<td>62101</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip to Freeze Sample</td>
			<td>Globe Scientific</td>
			<td>1411-10</td>
			<td>22x40mm, No. 1</td>
		</tr>
		<tr>
			<td>Coverslip to Seal Slide</td>
			<td>Globe Scientific</td>
			<td>1404-15</td>
			<td>22x22mm, No. 1.5</td>
		</tr>
		<tr>
			<td>DAPI Mounting Medium for Immunofluorescence</td>
			<td>Vector</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82029</td>
			<td>Duolink 1x Ligase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td>
		</tr>
		<tr>
			<td>Amplification red buffer</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82011</td>
			<td>Duolink 5x Amplification Red buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td>
		</tr>
		<tr>
			<td>Ligation Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82009</td>
			<td>Duolink 5x Ligation buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td>
		</tr>
		<tr>
			<td>Antibody Diluent</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82008</td>
			<td>Duolink antibody diluent,Comes with DUO92004 and DUO92002, Note: A 1x PBS/1% BSA solution can also be used as a substitute to dilute the antibody.</td>
		</tr>
		<tr>
			<td>Blocking Solution</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82007</td>
			<td>Duolink blocking solution, Comes with DUO92004 and DUO92002</td>
		</tr>
		<tr>
			<td>Mounting Medium for PLA</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td>Duolink In Situ mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>MINUS Probe</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td>Duolink In Situ Probe Anti-Mouse MINUS</td>
		</tr>
		<tr>
			<td>PLUS Probe</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td>Duolink In Situ Probe Anti-Rabbit PLUS</td>
		</tr>
		<tr>
			<td>Wash Buffer A</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82046</td>
			<td>Duolink In Situ wash Buffer A</td>
		</tr>
		<tr>
			<td>Wash Buffer B</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82048</td>
			<td>Duolink In Situ wash Buffer B</td>
		</tr>
		<tr>
			<td>Polymerase</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82030</td>
			<td>Duolink Polymerase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008</td>
		</tr>
		<tr>
			<td>Epifluorescent Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DFC300G camera, DM5500B microscope</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa 594</td>
			<td>JacksonImmuno</td>
			<td>115-585-146</td>
			<td>Use at 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 488</td>
			<td>JacksonImmuno</td>
			<td>111-545-144</td>
			<td>Use at 1:200</td>
		</tr>
		<tr>
			<td>Image Processing Software</td>
			<td>Adobe</td>
			<td></td>
			<td>Adobe Photoshop + Illsutrator CS3</td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>Corning</td>
			<td>7095B-5X</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>ACROS Organics</td>
			<td>187870100</td>
			<td>Prepare a 250mM working stock</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-FLAG</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td>Use at 1:1000 for immunofluorescence and PLA, pre-block with normal goat serum recommended</td>
		</tr>
		<tr>
			<td>Nailpolish</td>
			<td>L.A. colors</td>
			<td>CNP195</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Medium (NGM)</td>
			<td>various</td>
			<td></td>
			<td>See wormbook.org for protocol</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>JacksonImmuno</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Pasteur Pipette</td>
			<td>Globe Scientific</td>
			<td>135030</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1524</td>
			<td>Prepared as 0.1% stock solution in water, stored at -20C, and diluted 1:100 in water to coat slides</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Tritech</td>
			<td>PD7060</td>
			<td>60 mm diameter</td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP</td>
			<td>Thermo Fisher</td>
			<td>G10362</td>
			<td>Use at 1:200 for immunofluorescence, 1:4000 for PLA</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Thermo Fisher</td>
			<td>30-2066A-Brown</td>
			<td>Three-square 14x14mm autoclavable slides with bars are custom-ordered through Fisher Scientific. Poly-L-Lysine added to slides in the lab</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite solution</td>
			<td>Fisher Scientific</td>
			<td>SS290-1</td>
			<td></td>
		</tr>
		<tr>
			<td>task wipes</td>
			<td>Kimtech</td>
			<td>34120</td>
			<td>4.4x8.4 inch task wipes</td>
		</tr>
		<tr>
			<td>Trays (242x241x20mm)</td>
			<td>Thermo Fisher</td>
			<td>240845</td>
			<td>Used to make humid chamber</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ACROS Organics</td>
			<td>327372500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Milli-Q</td>
			<td></td>
			<td>Ultrapure water obtained from Milli-Q Integral Water Purification System</td>
		</tr>
		<tr>
			<td>Watchglass</td>
			<td>Carolina Biological</td>
			<td>742300</td>
			<td></td>
		</tr>
		<tr>
			<td>-20 &#176;C freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OP50 strain E. coli</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode strains used in this study (both available upon request)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mntSi13[pME4.1] II; unc-119(ed3) III; teIs1 [pRL475]</td>
			<td></td>
			<td>UMT 376</td>
			<td>dlc-1 prom::3xFLAG::dlc-1::dlc-1 3&#39;UTR; oma-1 prom::oma-1::GFP; Reference 24</td>
		</tr>
		<tr>
			<td>mntSi13[pME4.1] II; mntSi21[pXW6.22] unc-119(ed3) III</td>
			<td></td>
			<td>UMT 422</td>
			<td>dlc-1 prom::3xFLAG::dlc-1::dlc-1 3&#39;UTR; gld-1 prom::ceGFP::fbf-1 3&#39;UTR + unc-119(+); Reference: this study</td>
		</tr>
	</tbody>
</table>

in situ detection of ribonucleoprotein complex assembly in the c. elegans germline using proximity ligation assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

Co-immunostaining of both 3xFLAG::DLC-1; GFP and 3xFLAG::DLC-1; OMA-1::GFP germlines with FLAG and GFP antibodies revealed their patterns of expression in the germline (Figure 3Aii-iii,3Bii-iii). While GFP was expressed throughout the germline (Figure 3Aiii), OMA-1::GFP expression was restricted to the late pachytene and oocytes (Figure 3Biii)<sup cl...

Watch this Scientific Journal Video about In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay at JoVE.com

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Proximity ligation assay allows users to detect protein-protein interactions in situ, revealing spatial and temporal patterns of the interactions. This technique can help answer important questions about regulation of C.elegans development. This technique offers comparable sensitivity for detection of protein-protein interactions without the complexity of other techniques, such as FRET and BiFC.Individuals who have never dissected worms to release the gonads may struggle with this step. Practice dissecting worms, and use them for immunofluorescence to perfect the handling and dissection techniques before moving on to PLA. To begin, pick 30 to 40 young adult worms into a watch glass dish containing 500 microliters of 1x M9 and levamisole.After collecting the worms, carefully remove and discard most of the media to remove bacteria that is transferred along with the worms. Add in fresh 500 microliters of 1x M9 and levamisole, and use the pipette to gently draw up and dispense the media to rinse the worms. Carefully remove and discard most of the media.Repeat the wash two to three times. After the washes, leave the worms in about 100 microliters of media for no longer than eight minutes. Using a glass or polyethylene pipette, transfer the worms to a 25-millimeter-by-75-millimeter microscope slide coated with 001%poly-L-lysine.Remove the excess media so that approximately 15 microliters of media remains on the slide. Removal of excess buffer is critical to ensuring that the dissected worms and gonads will adhere to the slide. Under a dissecting microscope, with two 26 1/2-gauge needles, place one needle over the other so that the ends form a pair of scissors.Using the needles oriented in this fashion, cut the worms behind the pharynx to release the germlines. Dissect all the worms within five minutes. After all worms are dissected, gently place a 22-millimeter-by-40-millimeter coverslip over the slide so that it is perpendicular to the slide.The ends of the coverslip should hang off the slide. Freeze the slides on a pre-chilled aluminum block maintained on dry ice for at least 20 minutes. Gently place a chilled pencil on top of the coverslip to prevent the coverslip from becoming loose due to ice expansion.When ready for fixation, flick off the coverslips with a pencil, and immediately dip the slide into a jar containing fresh, ice-cold methanol for one minute. Then gently wipe the edges of the slide surrounding the sample, and apply 150 microliters of formaldehyde fixative at room temperature to fix for five minutes. Touch the slide to a paper towel at a perpendicular 90-degree angle to let the fixative run off the slide and absorb into the paper towel.Block the slides twice for 15 minutes at room temperature in a Coplin jar with 50 milliliters of PBT/BSA. Then block the slides with a PBT/BSA solution containing 10%normal goat serum. Gently wipe the edges that surround the slide, and apply 100 microliters of the solution to each slide.And gently agitate the slide to help spread the solution. Incubate for one hour at room temperature in a humid chamber. Place the slide on a paper towel to let the solution run off the slide, and gently wipe the edges.To block the slides, apply one drop of the blocking reagent to the 14-millimeter-by-14-millimeter space. Incubate the slides for one hour at 37 degrees Celsius in a humid chamber. Place the slides on a paper towel to let blocking reagent run off the slide, and gently wipe the edges.Use the antibody diluent to dilute the primary antibodies. Apply 40 microliters of the diluted primary antibody solution per 14-by-14-millimeter space on the slides. Place the slides in a humid chamber, and incubate overnight at four degrees Celsius.In the morning, wash the slides twice for five minutes, each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 rpm. Place the slides on a paper towel to let wash buffer run off the slide, and gently wipe the edges.Prepare a 40-microliter solution containing plus and minus probes. Add the solution to each 14-by-14-millimeter space. Incubate the slides in a humid chamber for one hour at 37 degrees Celsius.Wash the slides twice for five minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 rpm. Dilute the ligation buffer one to five with ultrapure water.Use this buffer to dilute the ligase one to 40 to prepare a working stock of ligation solution. Place the slide on a paper towel to let the wash buffer run off the sides, and gently wipe the edges. Add 40 microliters of the ligation solution to each 14-by-14-millimeter space.Incubate the slides in a humid chamber for 30 minutes at 37 degrees Celsius. After washing and drying the slides as previously described, add 40 microliters of freshly prepared amplification solution, protected from light, to each 14-by-14-millimeter space. Incubate the slides in the humid, dark chamber for one hour and 40 minutes at 37 degrees Celsius.Wash the slides twice for 10 minutes each with 50 milliliters of 1x wash buffer B, and wash the slides one time for one minute with 50 milliliters of 01x wash buffer B.After drying the epoxy-coated perimeter of the slide, add 10 microliters of mounting medium to each sample, and gently lay a coverslip on top, allowing for the mounting medium to spread out. Paint around the edge of coverslip with nail polish to seal the coverslip and slide. Avoid moving the coverslip.Let the nail polish harden for at least 20 minutes at room temperature, protected from light. Co-immunostaining of the germlines with FLAG and GFP antibodies revealed their pattern of expression in the germline. While GFP was expressed throughout the germline, OMA-1:GFP expression was restricted to the late pachytene and oocytes.FLAG immunostaining shows that 3xFLAG:DLC-1 was expressed throughout the germline in both strains. The region of interest for PLA quantification in the germline encompassed the late pachytene through the oocytes in all germlines. This is the region of OMA-1 expression.3xFLAG:DLC-1, OMA-1:GFP germlines appeared to have a greater quantity of PLA foci within this region compared to the 3xFLAG:DLC-1, GFP germlines. Ensure that there is enough water to raise the humidity in the chamber. Also, ensure that slides are level inside the chamber to prevent runoff of reagents.Following proximity ligation assay, the extruded gonads can be imaged on a confocal microscope and quantified using Fiji, ImageJ workflow. This quantification can help to determine if the interaction is statistically significant. PLA allowed us to assess in situ interactions between critical regulators of nematode germline development.

in-situ-detection-ribonucleoprotein-complex-assembly-c-elegans

Research

JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...