The author deeply thanks Dr. Asako Sugimoto (Tohoku University) for her helpful discussion. wyIs251 was a generous gift from Dr. Kang Shen (Stanford University). mnIs17 was provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by JSPS KAKENHI grant #17H05010 and #16H06536 and Daiichi Sankyo foundation, Brain Science Foundation and Naito Foundation.

1. Sample Preparation
<ol>
	<li>Generate a transgenic C. elegans strain of interest using transgenic methodologies18. Alternatively, obtain appropriate strains from the Caenorhabditis Genetics Center (CGC). To visualize axonal transport of synaptic vesicle precursors, use the transgenic line wyIs251 [Pmig-13::gfp::rab-3]7. To observe IFT, obtain the transgenic line mnIs17 [osm-6::gfp]19. These strains are used in this protocol. 
	NOTE: Either extrachromosomal array, genomic integration, or even GFP knock-in works. To reduce background signals, use cell-specific promoters, rather than pan-neuronal promoters. 
	NOTE: Maintenance of worms is described in other protocols20. Strains were maintained on lawns of Escherischia coli OP50 feeder on nematode growth medium (NGM) plates (1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) peptone, 1 mM CaCl2, 5 mg/mL cholesterol, 25 mM KH2PO4, 1 mM MgSO4 on 60 mm diameter plastic dishes) at 20 &#176;C.</li>
	<li>Grow worms at 20 &#176;C on NGM plates to any life cycle stage of the worms. 
	NOTE: One can observe both axonal transport and IFT at any stage. Late L4 to young adult is a good positive control because multiple publications have used these stages and visualized trafficking events14,21,22,23.</li>
</ol>
2. Preparation of 10% Agarose
NOTE: Many vendors provide similar products such as agar, agar powders, and agarose. Use electrophoresis grade agarose (gel strength &#62;1200 g/cm2). Cheap agar powders do not work because the resulting gel is not strong enough to immobilize worms.
<ol>
	<li>Mix 0.4 g agarose in 4 mL distilled water (DW) in a glass tube (1.5 cm x 10.5 cm). Put a lid on the glass tube to avoid evaporation.</li>
	<li>Put the glass tube on a 95 &#176;C heat block for 90 min. In addition, prepare another glass tube (1.5 cm x 10.5 cm) filled with DW and keep it at 95 &#176;C. Put a Pasteur pipet in the 95 &#176;C DW (Figure 1A). A lot of small bubbles will be seen in the agarose tube and the solution should be turbid. Leave it until the solution becomes clear. Then, mix by pipetting up and down until the agarose solution becomes homogeneous. 
	NOTE: Microwaves cannot be used to prepare 10% agarose solution when made from DW and agarose. Small bubbles caused by microwaves prevent agarose melting. However, solidified 10% agarose gel can easily be melted again using a microwave. Glass tubes containing 10% agarose gel can be prepared ahead of time and stored at 4 &#176;C for at least 6 months. If doing so, melt the stock using a microwave when needed and start from step 3 in this protocol. 
	NOTE: Agarose gradually loses the ability to solidify if incubated at 95 &#176;C for a long time or after repeated solidification and melting. If this happens, prepare new 10% agarose.</li>
</ol>
3. Preparation of Agarose Pad
<ol>
	<li>Using the warmed Pasteur pipet prepared in step 2.2, place 2 drops of 10% agarose on a 76 x 22 mm slide.</li>
	<li>Quickly cover with a second 76 x 22 mm2 slide before the agarose drop solidifies as shown in Figure 1B, and push down on the second slide to flatten the agarose solution. The thickness should be 0.5-1 mm.</li>
	<li>Quickly flush the Pasteur pipette by pipetting 95 &#176;C DW up and down until the agarose solution is washed out. Otherwise, the pipet will become clogged by agarose gel. Leave the pipette standing in the 95 &#176;C DW.</li>
	<li>Wait for 1 min. The agarose pad forms and becomes cloudy. Then, slide the slides apart by hand (Figure 1C).</li>
</ol>
4. Mounting of Worms
NOTE: Even small amounts of worm movement prevent good observation. Levamisole has traditionally been used to prevent worm movement on the agarose pad24,25. However, Levamisole inhibits neuronal receptors in C. elegans, and therefore may affect trafficking events in neurons15. Using polystyrene microbeads described hereis a good alternative17.
<ol>
	<li>Under a stereo microscope equipped with a slidable mirror transillumination, transfer ~30 transgenic worms expressing the appropriate markers to a new NGM plate without bacteria using a platinum wire pick. 
	NOTE: A platinum wire pick is made of platinum wire and Pasteur pipette (5 inches), and the tip of the platinum wire was flattened. The preparation of the platinum wire picks and handling of worms with these are described in the Wormbook (http://www.wormbook.org).</li>
	<li>Leave the plate for 1 min. Bacteria are automatically removed from the surfaces of worms as the worms move on NGM agarose gel without bacteria.</li>
	<li>Put a 0.5-1.0 &#181;L drop of 2.6% 100 nm diameter polystyrene microbeads in water onto the agar pad (Figure 1D). Transfer 10-20 worms from the bacteria-free NGM plate to the microbeads. Drop and spread the worms using the platinum wire pick to avoid the overlapping of worm bodies (Figure 1E).</li>
	<li>Apply a 22 x 40 mm2 coverslip (Figure 1F). Push gently to remove air bubbles, but avoid crushing worms. The sample is now ready for imaging. 
	NOTE: Because no anesthesia is administered, worms are fixed only by the frictional force generated by the agar pad, polystyrene microbeads, and coverslip17. The presence of feeder bacteria and/or too much solution will make the frictional force weaker. 
	NOTE: Different sizes of coverslips (e.g., 22 x 22 mm2, 18 x 18 mm2) work. However, larger coverslips can prevent immersion water or oil from the objective lens leaking into samples during the observation.</li>
</ol>
5. Observation
NOTE: Appropriate imaging parameters (laser power, gain, binning, etc.) will differ for each microscope system and camera. Here, a widefield microscope equipped with a spinning disk confocal scanner and a digital CCD camera is used.
<ol>
	<li>Set the temperature control of the stage to 20 &#176;C, or adjust the room temperature to 20 &#176;C.</li>
	<li>Put the sample slide on the microscope stage. Use 100x (numerical aperture = 1.3 or better) objective lens.</li>
	<li>Look for the areas indicated in Figure 2A (for wyIs251 and axonal transport) or Figure 2B (for mnIs17 and IFT) as positive controls. Other areas can be analyzed as well22.</li>
	<li>Set parameters (CCD Gain = 200, Binning = 1 or 2, laser power at 488 nm = 1.0-1.3 mW). Perform time lapse recording at 4 frames/s for up to 1 min. Save files as mutli-TIFF format. 
	NOTE: If GFP signals disappear and it is difficult to continue to observe GFP::RAB-3 or OSM-6::GFP for 30 s, the laser power is too strong. In that case, reduce the laser power. Conversely, if no vesicular movement is recorded, the laser power is too weak. In that case, increase the laser power.</li>
	<li>Observe a different worm and repeat 5.3 and 5.4. The observations can last for up to 20 min, but each worm should be recorded only once to avoid the effects of photo damage. 
	NOTE: Worms have been observed for at least 20 min without significant defects7,27,28. Longer observation may be possible but haven&#39;t been tested yet. A clear sign of neuronal death is the change of neuronal morphology such as neurite swelling. As weak GFP signals can be seen in axons and dendrites in both wyIs251 and mnIs17, neurite morphology can be easily checked by just looking at axons or dendrites. Neuronal morphology is shown in Figure 2.</li>
	<li>Generate movie files.
	<ol>
		<li>Open the multi-TIFF file using Fiji software. Go to File&#62;Open then select the multi-TIFF file saved in step 5.4. 
		NOTE: Fiji can be downloaded at jttps://fiji.sc. Image J and plugins can also be used to generate kymographs. If doing so, follow the relevant instructions for the chosen plugin.&#160;</li>
		<li>Click the &#34;Rectangular selection&#34; button (Figure 3A, arrow) and select the area of interest by drawing a rectangle with a computer mouse (yellow rectangle in Figure 3A). Go to Image&#62;Stacks&#62;Tools&#62;Make Substacks and select the slice numbers that are included in the movie. A substack is generated.</li>
		<li>Activate the substack window by clicking and go to Image&#62;Adjust&#62;Brightness/Contrast. A window to adjust the brightness and contrast shows up. In the window, slide the top bar (Minimum) to the right so that the background signal disappears and the second top bar (Maximum) to the left so that the moving vesicles and particles can be seen quite well over the background.</li>
		<li>Go to Image&#62;Stacks&#62;Time Stamper. As the movie is recorded at 4 frames/s in step 5.4, the time interval is 0.25 s. Thus, type &#34;0.25&#34; in Interval.</li>
		<li>Generate a movie file by selecting Save as&#62;AVI from the File menu (Movie 1 and 2). 
		NOTE: By using appropriate plugins, you can save the file as other formats such as &#34;mpeg4&#34; or &#34;mov&#34; files.</li>
	</ol>
	</li>
	<li>Generate kymographs.
	<ol>
		<li>Open the original movie files using FIji software again and repeat steps 5.6.2 and 5.6.3 to adjust the brightness and contrast.</li>
		<li>Click &#34;segmented line&#34; (Figure 3B, arrow). Using a mouse, draw a segmented line along the cilia or axon (yellow line, in Figure 3B).</li>
		<li>Go to Analyze&#62;Multi Kymograph&#62;Multi Kymograph (Figure 3C). Line width window appears (Figure 3D). Type &#34;3&#34; in the Linewidth window and Click OK (Figure 3D).</li>
		<li>Kymograph window is created. Save the image in TIFF or JPEG format.</li>
	</ol>
	</li>
</ol>

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The author has nothing to disclose.

Limitation with respect to existing methods 
The method described here is optimized to observe fast events such as axonal transport and IFT. Thus, immobilization is more prioritized than longer incubation. While we have been able to observe trafficking events for at least 20 min without significant perturbation, this method may not be always suitable to observe slow events requiring longer observations, such as axon elongation and cell migration. For longer observations, one needs to optimize the co...

Live cell imaging is an essential tool for analyzing intracellular transport. In neuronal cell biology, analyses of axonal transport with live cell imaging are essential for understanding neuronal function and morphogenesis1. Defects in axonal transport underlie several neurodegenerative disorders2. Kinesin superfamily proteins and dynein carry out axonal transport anterogradely and retrogradely, respectively1,2.
Cilia are another cellular compartment in which the microtubule network and trafficking machinery are highly developed3. Protein synthesis machinery is not localized in cilia, which means that ciliary proteins must be transported from the cytoplasm to the cilia tips. Cilia-specific kinesin and dynein, called kinesin-2 and cytoplasmic dynein-2, respectively, transport the components of cilia4, in a phenomenon called intraflagellar transport (IFT)5. Impairment of IFT causes a spectrum of diseases called ciliopathies6. Thus, analysis of the IFT mechanism by live cell imaging is required to understand basic mechanisms of ciliary formation and pathogenesis.
Caenorhabditis elegans (C. elegans) is a good model to study axonal transport and IFT7,8,9. To observe IFT, Chlamydomonas has been widely used as a model organism5,6. As Chlamydomonas is a unicellular organism, the relationship of IFT with aging, neuronal function, and behavior would be difficult to analyze. In addition, essential genetic techniques such as CRISPR/Cas9 have not been applied to Chlamydomonas. In higher model organisms, such as mice and Drosophila, disruption of axonal transport and IFT often causes lethal phenotypes because axonal transport and IFT are essential for the morphogenesis and homeostasis of the animals 10,11. In the case of mice, cell culture and transfection is generally needed to observe axonal transport and IFT, which requires many skills and extensive time12,13. In addition, a lot of important physiological context may be lost in cultured cells and cell lines. Because the nervous system is not essential for the survival of the worms, C. elegans mutants in which axonal transport or IFT are disrupted are often not lethal7,9,14. Axonal transport and IFT can be directly observed in vivo without dissection because C. elegans is transparent and it is therefore easy to observe GFP-tagged markers.
There are several protocols to immobilize C. elegans, such as using a microfluidic device15, agarose pads with anesthesia16, or microbeads17. The inclusion of anesthesia may inhibit the trafficking events in neurons15. A clear drawback of the microfluidic-device method is that preparing a microfluidic device is not always easy. Instead, immobilization by agarose pads and microbeads is a convenient and easy way to perform time lapse imaging in C. elegans. Here, I describe this basic protocol to immobilize C. elegans and visualize axonal transport and IFT in vivo in C. elegans. Compared to the other methods, the method described here does not require special equipment and is much cheaper and easier to perform.

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			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">C024401</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
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			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
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			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
</table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Axonal transport in DA9 neuron 
Using the wyIs251 line, both the anterograde and retrograde axonal transport of GFP::RAB-3 can be simultaneously recorded in the DA9 motor neuron. The average speed of anterograde and retrograde transport in the proximal dorsal axon of the DA9 neuron is about 1.8 and 2.6 &#956;m/s, respectively22. The number of moving vesicles is about 0.03 and 0.018 per &#956;m of axon per s. Thus, for a 30 s observati...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

immobilization-caenorhabditis-elegans-to-analyze-intracellular

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JoVE Journal

Developmental Biology

8.0K Görüntüleme.  Tohoku University. The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

Video: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

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 ...

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 ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

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 ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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 ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...