Work in the Arur Lab is supported by grants from the National Institutes of Health, NIHGM98200; Cancer Prevention Research Institute of Texas, CPRIT RP160023; the American Cancer Society Research Scholar Award (ACS RSG014-044-DDC); and by the Anna Fuller Funds.

The protocol described here is primarily for the invertebrate model system C. elegans, and follows all the ethical guidelines set forth by the institution.
1. Animal Maintenance
<ol>
	<li>Maintain C. elegans working stock cultures on Nematode Growth Medium20,21 (NGM, see Materials Table) agar seeded with E. coli OP50.</li>
	<li>Maintain worm stocks at temperatures between 16 &#176;C and 25 &#176;C. 
	NOTE: Culturing worms at higher temperatures results in faster growth rate, often, aberrant germline development and lower brood sizes. Additionally, temperature sensitive genotypes will display different phenotypes at different temperatures.</li>
	<li>Transfer worms to new NGM plates every 2 - 3 days depending on their growth rate so as to maintain a constant supply of well-fed worms. 
	NOTE: For any given experiment involving dpERK levels, worms should NOT be obtained from a starved or crowded plate. Crowding or starvation impacts insulin signaling in worms22, and insulin signaling regulates ERK12 activity. Thus worms from starved or overcrowded plates will result in variable and difficult-to-reproduce staining patterns.</li>
</ol>
2. Dissection of Adult Worms for Obtaining Gonads
<ol>
	<li>Pick 100 - 150 WT (N2) or desired genotype of worms at the desired and specific developmental stage (L1, L2, L3, L4, adult, etc.) in 100 &#181;L of M9 buffer in a 1.5 mL microcentrifuge tube.</li>
	<li>Fill the microcentrifuge tube with 900 &#181;L of M9 buffer (see Materials Table). Centrifuge the tubes at 1,000 x g in a microcentrifuge for 1 min at ambient temperature.</li>
	<li>Gently remove 900 &#181;L of the M9 buffer and discard. Remove the buffer under a dissecting microscope to ensure that the worms are not accidently removed.</li>
	<li>Repeat steps 2.2 - 2.3 two more times with fresh M9 buffer each time. This ensures that any bacteria that were carried over with the worms are effectively washed away.</li>
	<li>After the final wash remove 900 &#181;L of M9 buffer from the tube and discard.</li>
	<li>Transfer the remaining 100 &#181;L of M9 buffer containing the 100 - 150 worms with a 200&#160;&#181;L-micropipette tip to a flat bottom glass watch dish. Ensure that the micropipette tip is cut at the 10&#160;&#181;L mark before use. Not cutting the tip will result in shearing of the worms.</li>
	<li>Add 1 - 3 &#181;L of 0.1 M levamisole to the glass watch dish containing the worms in M9 buffer. Gently swirl the levamisole and the M9 to enable even mixing until the worms stop moving. 
	NOTE: Levamisole stocks can be stored at -20 &#176;C for long-term storage. Here, use the higher concentration of 1 - 3 mM than what has been described in literature23 of 0.2 - 0.25 mM of levamisole in order to obtain rapid loss of mobility in the animals. If the animals flay around for too long in the liquid suspension, the resulting patterns of dpERK in the gonads can be variable (due to stress or lack of nutrition or both). More reproducible staining patterns have been achieved with 1 - 3 mM levamisole for dissection.</li>
	<li>Attach two 25 G needles to two 1 mL syringes (the size of the syringe does not matter, the gauge of the needle is critical). Position one syringe in each hand (Figure 2).</li>
	<li>Under a dissecting microscope, position each needle under and over each worm as shown in Figure 2 and place a fine cut on the worm near the second pharyngeal bulb in a scissor-like motion. After one cut in the worm move on to the next animal until all the animals in the dish have been cut at least once. 
	NOTE: Be mindful that steps 2.8 - 2.9 are time sensitive. Dissect all worms in the dish in under five min for a reproducible dpERK pattern. Longer dissection times will result in a turning off of the dpERK signal, especially in Zone 1. Adjust the number of worms per round of dissection (i.e., 50 worms vs.&#160;150) if necessary to stay in the allotted time frame.
	<ol>
		<li>In case an extruded gonad is not visible during the dissection, do not attempt another cut in the same animal. Usually that will only shear the animal and not result in extrusion of the gonad. Instead, move on to the next animal. Worms where the gonads do not extrude will appear as such on the slides that will be made in section 6 and can be ignored during imaging 
		NOTE: Through all of the dissection and processing steps, it is important to bear in mind that the gonad will remain attached to the body/carcass. Do not force the gonad and the body apart with the needle. Often times an aberrant tear in the gonadal tissue in an attempt to sever the gonad from the body can result in spurious antibody signal. The body/carcass can be ignored during the imaging steps (section 6), and only the gonad imaged. Additionally, sometimes the intestinal cells can also serve as internal controls/somatic controls for the antibody being assayed.</li>
	</ol>
	</li>
</ol>
3. Gonad Fixation
<ol>
	<li>After the dissection is complete, add 2 mL of 3% paraformaldehyde (PFA) directly to the glass watch dish containing the 100 &#956;L of M9 and the dissected animals and cover with Parafilm. Place the covered watch glass dish in a chemical fume hood. 
	Caution: PFA is a hazardous chemical. Thus, the fixation should be performed in the chemical fume hood.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Using a disposable 9&#34; glass Pasteur pipette add 3 mL of 1x PBS-T (see Materials Table) to the watch glass dish containing the dissected animals. Mix by drawing the PFA solution and the 1x PBS-T with the dissected animals into the Pasteur pipette and releasing gently back into the watch glass dish. Do not vortex the tubes during the washes.</li>
	<li>Using a fresh glass Pasteur pipette draw the 5 mL of liquid (containing dissected worms, PFA and 1x PBS-T) from the watch glass dish into the Pasteur pipette and transfer the contents into a fresh 5 mL glass conical tube.</li>
	<li>Centrifuge the conical tubes for 30 sec in a clinical centrifuge at 1,000 x g. Use glass Pasteur pipettes and conical tubes as dissected gonads stick to plastic.</li>
	<li>Remove and discard the supernatant taking care so as not to disturb the dissected animals. Remove the liquid after each wash from the conical tube under a dissecting microscope so as not to affect the dissected gonads, and minimize their loss.</li>
	<li>Using a fresh Pasteur pipette, add 5 mL of 1x PBS-T to the conical tubes. Centrifuge the conical tubes in a clinical centrifuge for 30 s at 1,000 x g. Remove and discard the supernatant taking care so as not to disturb the dissected animals.</li>
	<li>Repeat step 3.7 for a total of three times.</li>
	<li>Using a fresh Pasteur pipette, add 2 mL of 100% methanol to the tubes, and gently mix the gonads with the methanol by drawing up into the Pasteur pipette. Incubate the tube at -20 &#176;C for a minimum of 1 h. 
	NOTE: Dissected gonads can be stored in methanol for up to 2 days for dpERK staining. Storage for more than 2 days and up to 2 weeks is compatible with other antibody staining applications, such as anti-Lamin antibody, but not with the dpERK antibody.</li>
	<li>After the desired incubation time, repeat step 3.7 for a total of three times in order to wash the gonads. Do not vortex the tubes during the washes. After the final wash leave 500 &#956;L of 1x PBS-T in the 5 mL conical tube.</li>
	<li>Using a fresh Pasteur pipette transfer the contents of the conical glass tube into a fresh 1 mL glass tube (6 mm x 50 mm). Allow the dissected animals to settle by gravity for 5 - 10 min.</li>
	<li>Using a fresh Pasteur pipette and under a dissecting microscope, remove as much of the wash as possible from the 1 mL glass tubes without disturbing the dissected gonads.</li>
</ol>
4. Blocking and Primary Antibody Treatment
<ol>
	<li>Add 100 &#181;L of 30% Normal Goat Serum (NGS) diluted in 1x PBS-T (see Materials Table) to the dissected animals in the 1 mL glass tubes. 30% NGS serves as the blocking buffer. Cover the tube with Parafilm to avoid evaporation of the liquid. Incubate at RT for 1 h or at 4 &#176;C overnight.</li>
	<li>After the desired time of incubation, remove the blocking buffer using a fresh Pasteur pipette, removing as much liquid as possible. Use a dissecting microscope to ensure that gonads are not drawn up in the Pasteur pipette.</li>
	<li>Dilute the MAPKYT antibody (see&#160;Materials Table, referred to in this method as dpERK) at 1:400 in 30% NGS. Prepare enough antibody dilution to use 100 &#181;L per tube. Unused, diluted antibody can be stored at 4 &#176;C for a week.</li>
	<li>Add 100 &#181;L of the diluted anti-dpERK antibody solution to the 1 mL glass tubes containing the dissected animals. Seal the tubes with Parafilm. Incubate the tubes at 4 &#176;C overnight.</li>
	<li>After overnight incubation, add 800 &#181;L of 1x PBST to the tubes at RT.</li>
	<li>Draw the sample up in a fresh glass Pasteur pipette and release gently to ensure that the gonads are evenly suspended in the 1x PBS-T. Let the gonads settle to the bottom with gravity. Remove the supernatant. This ensures that the dissected gonads are washed but not damaged during the process.</li>
	<li>Repeat steps 4.5 - 4.6 for a total of three washes. Do not vortex the tubes during the washes. After the third wash be sure to remove and discard as much supernatant as possible without disturbing the dissected animals. Ensure that a fresh Pasteur pipette is used each time.</li>
</ol>
5. Secondary Antibody Treatment
<ol>
	<li>During the primary antibody washes prepare the dilution for the secondary antibody. Dilute the anti-mouse secondary antibody at 1:500 in 30% NGS. As with the primary antibody, use 100 &#181;L per tube. Unused antibody can be stored at 4 &#176;C for a week.</li>
	<li>Add 100 &#181;L secondary antibody solution to the 1 mL glass tubes containing the dissected animals. Cover each tube with Parafilm, and then wrap the tubes in aluminum foil to keep the contents of the tube in dark. Incubate the tubes at RT for 2 h, or alternatively at 4 &#176;C overnight.</li>
	<li>After the desired time of incubation, add 800 &#181;L of 1x PBS-T to the tubes and repeat steps 4.5 - 4.6 for a total of three times. After the final wash, remove and discard as much of the supernatant as possible under a dissecting microscope using a fresh Pasteur pipette.</li>
	<li>Prepare the DAPI solution during the washes for the secondary antibody. Dilute 1 &#181;L of DAPI (from a 1,000x stock of 1 mg/mL stored at -20 &#176;C) in- 1 mL of 1x PBST.</li>
	<li>Add 800 &#181;L of the diluted DAPI solution to the tubes containing the dissected animals. Cover the mouth of the tube with Parafilm, and wrap each tube in aluminum foil. Incubate at RT&#160;for 20 min.</li>
	<li>After the incubation with DAPI, remove as much of the DAPI solution as possible with a fresh Pasteur pipette, under a dissecting microscope so as not to disturb the dissected animals.</li>
	<li>Add 1 drop of mounting solution to the tubes. Wrap each tube in aluminum foil. 
	NOTE: For visualizing dpERK staining, stained gonads should be mounted for microscopy immediately. For other antibody applications such as phosphorylated serine 10 Histone H3, gonads can be kept in mounting media for up to 2 weeks at 4 &#176;C in the dark.</li>
</ol>
6. Assembling Slides and Imaging
<ol>
	<li>Place a layer of lab tape lengthwise, on top, of two glass microscope slides (25 mm x 75 mm x 1 mm) as seen in Figure 3. Place a fresh clean glass microscope slide in between the two taped slides. 
	NOTE: The thickness of the tape helps generate an agarose pad on the middle slide. Thickness of the agarose pad is almost equal to that of the tape, as the tape serves as a spacer for creating the agarose pad.</li>
	<li>Drop ~ 200 &#181;L of melted 2% agarose (made in distilled water) on the microscope slide in the middle. Quickly place a fresh clean slide, perpendicular to and on top, of the agarose.&#160;</li>
	<li>Let the agarose solidify (1 - 2 min).</li>
	<li>Remove the top microscope slide very gently, such that the agarose pad is not destroyed. The agarose pad can remain attached either to the top or the bottom slide. It does not matter which slide the agarose pad remains attached to, as long as it is a smooth surface of agarose.</li>
	<li>Using a Pasteur pipette transfer the dissected animals onto the agarose pad. Remove any excess liquid from the slide using a Pasteur pipette under a dissecting microscope.</li>
	<li>Using an eyelash hair or finely drawn capillary, push the gonads and intestines away from one another in order to spread the gonads out on the slide.</li>
	<li>Cover the microscope slide with a 24 mm x 50 mm size coverslip. Take care to ensure that the coverslip is lowered gently onto the slide with minimal air bubbles being formed between the coverslip and the slide.</li>
	<li>Store at 4 &#176;C overnight in a slide box (an opaque slide box with the slides lying flat, coverslip up) to allow excess liquid to evaporate and gonads to become somewhat flattened (due to the weight of the coverslip on the gonads). The slight flattening of the gonads allows for better imaging of an otherwise round gonadal tube.
	<ol>
		<li>Once the coverslip is mounted, do not to apply pressure to the top of the coverslip with fingers. Often this results in distortion of gonads and loss of the dpERK signal.</li>
		<li>Flatten the gonads overnight&#160;only for more effective imaging. The slides are ready to be viewed as soon as they are assembled. To take images immediately, gently absorb the excess liquid between the coverslip and the slide from the corners using a clean lab tissue.</li>
	</ol>
	</li>
	<li>After the overnight&#160;period, seal the slide and coverslip with transparent nail polish topcoat on the four corners of the coverslip and the slide boundary. The nail polish/coverslip seal ensures that the coverslip does not move while imaging and protects against destruction of the samples.</li>
	<li>Image the gonads with a compound microscope at 63X. However the objective lens and microscope can be varied based on the availability of microscopes and user application. Because the size of the entire gonad from the proliferative region to the oocytes is larger than can be captured in one image, the images are taken as a montage with overlapping boundaries as shown in Figure 4.11-14, 18.
	<ol>
		<li>In many cases, edit the carcass after the final imaging and assembly, as long as no major alterations are made to the germline image. Store the raw images along the assembled image to enable comparison of the &#39;before&#39; assembly/editing and &#39;after&#39; assembly/ editing of the images. 
		NOTE: The microscopy images must not be altered for signal strength or saturation during the assembly of the montage.</li>
	</ol>
	</li>
</ol>

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The authors declared that no competing interests exist.

Active ERK (dpERK) follows a stereotypical spatial and dynamic localization pattern in the C. elegans germline. This stereotyped dpERK spatial pattern (Figure 5), and amplitude in an adult C. elegans gonad, can be effectively correlated with the many biological processes that ERK regulates. For example, an inability to activate ERK in Zone 2 results in an arrest in oocytes in prophase of meiosis, a phenotype observed in female germlines that do not specify sperm, and thus do not activate ERK in mature oocytes (Figure 5). Mating with males&#39; results in effective accumulation of dpERK in Zone 2 in the female germlines11,13, coupled with onset of oocyte maturation and ovulation. Thus analysis of spatial patterns and temporal activation of dpERK upon certain genetic perturbations (such as RNA interference mediated gene inactivation) or chemical treatments allows for identification of factors that regulate ERK signaling and thus ERK-dependent biological processes. Such analyses also enable discovery of novel genetic interactions between signaling pathways.
A critical bottleneck for any imaging based analysis is the availability of functional reagents such as antibodies that are specific to the application. If such a reagent exists then methods such as the one described above can be easily adapted for the analysis of localization pattern for any protein of interest. The application is thus limited by the availability of a functioning antibody. Additionally, because the method describes dissection and staining at a given developmental time, it only enables the capturing of information in one time frame, and does not shed light on the dynamics or protein regulation in real time or rate of protein turnover.
The method described above is adapted from Francis et al.23, which is distinct from the Seydoux and Dunn method24 for gonad extrusion and antibody staining. The method based on Francis et al. will be called the &#34;suspension method&#34; and the Seydoux and Dunn method called the &#34;freeze cracking method&#34; for comparison. The suspension method has been very effective in our hands for obtaining reproducible localization patterns of signaling molecules such as dpERK, that are inherently more sensitive to harsh handling conditions. In the case of dpERK localization, in our experience, the signal in Zone 1 is virtually undetectable with the freeze cracking method. Additionally, sample drying, which can often occur with the freeze cracking method, results in spurious antibody signals. The suspension method minimizes sample drying, since the gonads are suspended in liquid throughout the process. We also find that the suspension method is useful for imaging multiple different genotypes on a single slide. For example, if two genotypes, such as hermaphrodites vs females are to be directly compared for dpERK localization, the two genotypes can be mixed together and processed. This is possible because the phenotypes are distinct and can be distinguished via DAPI morphologies. Staining in the same tube followed by imaging on the same slide allows for direct comparison between the gonads from different genotypes. Alternatively, if the DAPI morphologies are not distinguishable, then a two-step antibody staining can be adopted. First each individual genotype is treated with two distinct antibodies, Antibody A for WT and Antibody B for mutant (both antibodies need to be raised in a host distinct from the dpERK antibody). Once the two genotypes have been stained with the primary antibody, and washed, they can be mixed together in one tube and now stained with the dpERK antibody, followed by a secondary antibody treatment to visualize all the antibodies in the tube. An ability to compare different genotypes on a single slide, especially when deductions of activation patterns are being made ensures accurate interpretations not affected by distinct staining conditions.
While advantageous, there are multiple steps throughout the suspension method that require careful manipulation. It is critical that the dissections occur in a time efficient manner; importantly, the dissections should not take over 5 min. Longer dissection times result in variable dpERK signal, which can often be misinterpreted as regulated changes in ERK activation, rather than as technical failures. It is crucial to start with over 100 - 150 animals for each dissection because throughout the various wash steps the gonads can easily be lost from the tubes due to pipetting errors. Loss of gonads can be reduced either by dissecting in multiple small batches (such as three batches of 50 animals each) that can be combined, or by dissecting one large batch of 150. Another challenge is getting the gonads to lie in a well-spaced formation on the slide so that the entire distal gonad can be imaged. To enable this, use an eyelash on a matchstick or a pulled pipette to space out the gonads. However, care should be taken so as not to damage the gonads during this process. Often with these techniques it takes multiple attempts to master the dissections as well as arrangements of the gonads onto the slides.
Once this technique has been mastered, it serves as a powerful method for varied biological analyses, and can be used to study more than just dpERK levels. For example this method can be used to visualize active p38 levels, or active CDK levels, or any protein for which an antibody reagent exists. Additionally, the method can be coupled with a chemical inhibition screen in whole animals to assay for novel inhibitors or activators of the pathway, via assaying for dpERK levels. This will allow for an in vivo readout of both reaction and sensitivity to any inhibitors that may interact with the RTK-RAS-ERK signaling pathway. Moreover, this method can be used in antibody combinations with multiple signaling outputs that can all be used and visualized at one time. Using multiple antibodies allows correlation between multiple pathways, their activation status, and outcomes all within the same tissue, enabling better understanding of these interactions. These are just a few examples of further applications of this method, but this system can be modified to study multiple research interests.

The Receptor Tyrosine Kinase (RTK)-RAt Sarcoma (RAS)-Extracellular signal Regulated Kinase (ERK) pathway relays extracellular signals through a conserved kinase cascade that results in the phosphorylation and activation of ERK1-3. ERK proteins are members of the conserved proline-directed serine/threonine MAP (Mitogen Activated Protein) kinase family, and are directly activated by MEK via dual phosphorylation on the threonine (T) and tyrosine (Y) of the conserved TEY motif. Active ERK (referred to as diphosphorylated ERK, or dpERK) then regulates many cellular and developmental processes through its phosphorylation of a battery of downstream substrates1-3. Thus abnormal ERK activity leads to many cell and developmental defects4-7.
Stringent regulation of ERK activity is critical for normal development: in mammalian systems too much ERK activity leads to excessive cellular proliferation leading to oncogenic growth; too little activity leads to cell death4,6. Additionally, changes in the duration of ERK activity can also lead to distinct outcomes: in PC12 cells, ERK activation for 30 min or less induces cell proliferation, but ERK activation for 60 min or more induces neuronal differentiation8,9. Tight regulation of ERK activity is thus clearly essential for normal development and homeostasis.
C. elegans is a powerful, and genetically malleable model system to dissect the function and regulation of the RTK-RAS-ERK signaling pathway3,10-15. Relative to mammalian systems, which contain multiple genes for RAS and ERK, C. elegans contains one RAS gene (let-60) and one ERK gene (mpk-1), rendering it a genetically more facile system in which to dissect the function of this pathway3,10-14. The C. elegans germline is essentially a tube that consists of mitotic stem cells at its distal end and mature oocytes at its proximal end (Figure 1)16. Germ cells initiate meiosis just proximal to the distal mitotic zone, and progress through an extended meiotic prophase (pachytene), after which they begin to form oocytes in the loop region, finally undergoing oocyte maturation in the proximal region16.
Genetic studies from multiple labs, including our own, have shown that the RTK-RAS-ERK pathway is essential for germline development in C. elegans12,13,15,17-19. Specifically, we found that ERK controls and coordinates at least nine distinct biological processes during germline development, ranging from developmental switches such as germ cell apoptosis to cell biological processes such as oocyte growth11,18. Much like in mammalian systems, too much ERK activity in the C. elegans germline results in production of multiple small oocytes, while too little activity results in one large oocyte11. Thus tight regulation of dpERK is essential for normal germline development. The active form of ERK, as visualized by an antibody specific to dpERK, displays a stereotypical, dynamic, bimodal localization pattern: dpERK is high during mid-pachytene (Zone 1, Figure 1), low in the loop region and high again in mature oocytes (Zone 2, Figure 1). Recently, we found that nutrition acts through the Insulin-like Growth Factor receptor-1 (daf-2) to activate ERK in Zone 112; prior work showed that the sperm signal (via the Ephrin receptor tyrosine kinase) activates ERK in Zone 213.
Given that active ERK functions as a rheostat in the germline to regulate oocyte growth,&#160;spatial and temporal localization, as well as amplitude of dpERK, is key to understanding its normal signaling outcome. Using the method described here, changes in the stereotypical localization of dpERK can be easily monitored and predictions derived on the impact of the environmental or genetic perturbations on ERK activity and thus function. Thus, assaying for dpERK enables a comprehensive understanding of its role during germline development.

<table border="0" cellpadding="0" cellspacing="0" width="749">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Agarose</td>
			<td style="width:159px;">Sigma Inc.</td>
			<td style="width:119px;">A9539</td>
			<td style="width:255px;">Dissolve 2 g in 100 ml to make the 2% agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Flat bottom glass watch dish</td>
			<td style="width:159px;">Agar Scientific</td>
			<td style="width:119px;">AGL4161</td>
			<td style="width:255px;">We use glass, because dissected gonads often stick to plastic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">25 gauge needles</td>
			<td style="width:159px;">BD PrecisionGlide</td>
			<td style="width:119px;">305122</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Syringes (could be 1 or 5 ml)</td>
			<td style="width:159px;">BD Syringes</td>
			<td style="width:119px;">1 ml: BD 309659</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscope slides (25mm x 75mm x 1.0mm)</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:119px;">12-550-343</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Coverslips (24x50mm)</td>
			<td style="width:159px;">Corning</td>
			<td style="width:119px;">2935-245</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">5ml glass conical tube</td>
			<td style="width:159px;">Corning</td>
			<td style="width:119px;">8060-5</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">9&#34; disposable Pasteur pipette</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:119px;">13-678-20D</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Clinical bench top centrifuge</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glass&#160; tubes (6x50mm)</td>
			<td style="width:159px;">Fisherbrand</td>
			<td style="width:119px;">14-958-A</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">*M9 solution</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Levamisole</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">L9756</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">3% Paraformaldehyde</td>
			<td style="width:159px;">Electron microscopy services</td>
			<td style="width:119px;">17500</td>
			<td style="width:255px;">Obtained as 16% solutions in ampoules, and diluted to 3% in Potassium Phosphate Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">**PBS</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1X PBST</td>
			<td style="width:159px;">1X PBS with 0.1% Tween 20&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Methanol</td>
			<td style="width:159px;">Electron microscopy services</td>
			<td>18510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Normal goat serum (NGS)</td>
			<td style="width:159px;">Cell Signaling</td>
			<td style="width:119px;">5425</td>
			<td style="width:255px;">Diluted to 30% in 1x PBST</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MAPKYT (dpERK) antibody&#160;</td>
			<td style="width:159px;">Sigma Inc.</td>
			<td style="width:119px;">M9692</td>
			<td style="width:255px;">Dilution 1:400 in 30% NGS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Secondary antibody</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:119px;">A-11005</td>
			<td style="width:255px;">Dilution 1:500 in 30% NGS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DAPI</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">D9542</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vectashield</td>
			<td style="width:159px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">*M9 Buffer Recipe</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">**PBS (1X)</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td>8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 0.133 g CaCl2.2H2O, 0.10 g MgCl2.6H2O, H2O to 1 litre</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:216px;">Nematode Growth Medium (NGM)</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;">3 g NaCl, 17 g Agar, 2.5 g Peptone, 975 mL of water in 2 L Erlenmayer Flask. Autoclave for 50 minutes. Cool, and add 1 mL of 1M CaCl2, 1 mL of 5 mg/mL cholesterol, 1 mL of 1M MgSO4 and 25 mL of 1M KPO4.&#160;</td>
		</tr>
	</tbody>
</table>

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.

In WT adult hermaphroditic animals, dpERK is typically visualized in mid-pachytene region, Zone 1, and in the most mature oocytes from -1 through -4 or -5 (Zone 2). Perturbations in this activation pattern reflect changes to the signaling pathway. Female germlines do not specify sperm, and thus do not display activation of ERK in Zone 2, with only weak activation in Zone 1. These are represented in Figure 5.
<img alt="Figure 1" src="/files/ftp_upload/54901/54901fig1.jpg" /> 
Figure 1: C. elegans&#160;germline Morphology. Differential Interference contrast (DIC) image of an adult hermaphrodite with one U-shaped gonad arm outlined with the white line. The adult hermaphroditic gonad contains mitotic cells at the distal tip. The mitotic cells enter meiosis and progress through meiotic prophase (pachytene) developing into mature oocytes in the proximal gonad. Zone 1 and Zone 2 mark stereotypical localization of dpERK in an adult hermaphroditic gonad. Scale: 20 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54901/54901fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54901/54901fig2.jpg" /> 
Figure 2: Demonstration of Needle Positions during dissections. Left: Photograph of a dissection in process. Right: Needle positions with reference to the worm. <a href="http://cloudflare.jove.com/files/ftp_upload/54901/54901fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54901/54901fig3.jpg" /> 
Figure 3: Demonstration of Agarose Slide Preparation. (A) Place a tape lengthwise across 2 clean microscope slides. Place a fresh clean microscope slide between the two slides with the tape as shown. The three slides are next to each other lengthwise. (B) Add melted agarose to the slide in the center. (C) Place a fresh microscope slide perpendicular to, and on top of, the middle slide, carrying the drop of agarose. (D) Allow the agarose to solidify. (E) Remove the top slide leaving behind the solidified agarose pad. Use the bottom slide with the agarose pad for mounting dissected and stained germlines. <a href="http://cloudflare.jove.com/files/ftp_upload/54901/54901fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54901/54901fig4.jpg" /> 
Figure 4: Imaging the Slides as a Montage. Montage of a wild type germline with DAPI (top) and dpERK (bottom) channel. Images taken at 63X with overlapping boundaries, white boxes for panel A and B and green boxes for panel B and C. The DAPI and dpERK images for each panel were taken simultaneously in two different channels. The assembled image is shown in Figure 5A. Scale bar: 20 &#956;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54901/54901fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54901/54901fig5.jpg" /> 
Figure 5: Dynamic Temporal/Spatial Activation of ERK. (A-C) WT&#160;(N2) adult (24 hours past mid-L4 stage of development) hermaphrodite germlines stained for DNA (B, DAPI, white) and dpERK (C, red). The dpERK signal in Zone 1, the pachytene region, and Zone 2, the mature oocytes is highlighted. (D-F) fog-2(oz40) female germlines (at 8 h&#160;past mid-L4 stage of development) stained for DNA (E, DAPI) and dpERK (F). Young female germlines display weak dpERK in Zone 1, and in a single oocyte in a sperm independent manner. Zone 2 is sperm dependent, and thus absent in the female germline. Scale bar: 20 &#956;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54901/54901fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Spatial and Temporal Analysis of Active ERK in the C. elegans Germline at JoVE.com

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline | Developmental Biology | Video

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.

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 ...

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10.1K Views.  Baylor College of Medicine. We present an immunofluorescence 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.

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

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 ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

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 ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

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 ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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 ...