We thank Monash Microimaging for their technical support. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by a Monash University Biomedicine Discovery Fellowship, NHMRC Project Grant (GNT1105374), NHMRC Senior Research Fellowship (GNT1137645) and veski innovation fellowship: VIF 23 to Roger Pocock.

1. Preparation and Worm Husbandry
Note: Refer Table of Materials for all product information.
<ol>
	<li>OP50 Escherichia coli culture: culture OP50 bacteria in Lysogeny broth (LB) (1% tryptone, 0.5% yeast, 0.5% NaCl, pH 7.0) overnight at 37 &#176;C without antibiotics.</li>
	<li>Seed 400 &#181;L of OP50 bacteria to nematode growth media (NGM) plates (1.5 g of NaCl, 8.5 g of agar, 1.25 g of peptone, 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in ethanol, 1 mL of 1 M MgSO4, and 25 mL of 1 M KPO4 buffer) and air dry the bacteria for 48 h.</li>
	<li>Pick worms on seeded NGM plates and incubate at 15 &#176;C or 20 &#176;C.
	<ol>
		<li>For the analysis of the adult hermaphrodite germline, pick well-fed L4 worms to the bacterial lawn and incubate overnight at 20 &#176;C.</li>
		<li>For scoring sperm number in hermaphrodites, incubate L4 worms at 20 &#176;C for 12 h until the worms reach the L4/adult molt. To synchronize the worms to the L4 stage, prepare eggs by picking adult worms with a lot of eggs in bleach solution (1 M NaOH and bleach in 1:1 ratio). Allow the eggs to hatch and pick L4 animals for the experiment.</li>
	</ol>
	</li>
	<li>Prepare Teflon microscope slides by placing 25 &#181;L of poly-L-lysine solution on the slide and spreading the solution well, using a pipette tip. After removing excess poly-L-lysine with paper towel, allow the slides to air dry and incubate the slides at 65 &#176;C for 15 - 20 min.</li>
</ol>
2. Germline Dissection and Staining6,13
<ol>
	<li>Spot 10 &#181;L of 0.01% tetramisole (anesthetic) on a 22 mm &#215; 22 mm coverslip and pick one-day old adult worms into it.</li>
	<li>Dissect the worm at the tail as it becomes sedated using a syringe (5 mL) and a needle (24&#34; x&#160;1&#34;), and ensure that the germline is not damaged. 
	NOTE: The dissection must be carried out before the worm becomes fully paralyzed so that the negative pressure in the worm and the worm movement aid the ejection of the germline. For scoring sperm, precautions must be taken not to damage the spermatheca by the needle during dissection. Avoid touching the germline with the needle except the dissection point after the spermatheca. If there is break in the germline compartment or any discharge from the germline is observed, the germline should not be used for analysis.</li>
	<li>Place the inverted coverslip on a poly-L-lysine coated slide, keeping the dissected germline on the slide.</li>
	<li>Remove excess liquid under the coverslip by placing a paper tower at a corner of the coverslip, then place the slide in liquid nitrogen for 1 min. Quickly remove the coverslip using a needle with the germlines on the slide.</li>
	<li>Transfer the slides into -20 &#176;C methanol for 30 - 60 s in a chamber, then incubate for 30 min in freshly prepared fixing solution (1x phosphate buffered saline (PBS) containing 0.08 M HEPES pH 6.9, 1.6 mM MgSO4, 0.8 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), and 3.7% paraformaldehyde) at room temperature.</li>
	<li>Wash the slides by placing in a chamber containing 1x PBS, pH 7.4 with 0.1% Tween-20 for 10 min. Repeat this step once.</li>
	<li>Block the germlines with 30 &#181;L of blocking solution (30% of goat serum prepared by adding 900 &#181;L of goat serum in 1700 &#181;L of distilled H2O and 300 &#181;L of 10x PBS) in a humid chamber prepared by placing wet paper towel in a plastic box at room temperature for 30 min.</li>
	<li>Add 50 &#181;L of REC-8 antibody solution prepared in 30% goat serum in a ratio 1:300 to each sample. Incubate overnight at 4&#176;C.</li>
	<li>Wash the slides by placing in a chamber containing 1x PBS with 0.1% Tween-20 for 10 min. Repeat the step once and remove excess liquid by placing a paper towel at the corner of the slide.</li>
	<li>Prepare secondary antibody solution by diluting green fluorophore (488 nm emission wavelength) conjugated secondary antibody (1:1000), phalloidin (actin stain, 1:4000), and DAPI (DNA stain, 1:4000) in 30% normal goat serum.</li>
	<li>Add 50 &#181;L of secondary antibody solution to the slide.</li>
	<li>Incubate for 1 h at room temperature.</li>
	<li>Wash the slides twice by placing in a chamber containing 1x PBS with 0.1% Tween-20 for 10 min. Wipe off excess liquid.</li>
	<li>Add 30 &#181;L of fixing reagent onto the slide and place a 12 mm2 x 1.5 &#181;m coverslip on top and dry the slides at 4 &#176;C overnight.</li>
</ol>
3. Confocal Microscopy
<ol>
	<li>Turn on the confocal microscope, lasers, resonant scanner, and confocal microscope software. Place the slides with stained germlines on the slide holder above the 63X objective.</li>
	<li>Locate a germline, focus on the top of the germline and mark it using confocal microscope software. Similarly focus on the bottom of the germline and mark it to establish the total germline thickness.</li>
	<li>Image the entire germline by defining the thickness of each slice up to 0.5 &#181;m. Mark the complete germline and start acquisition. Scan each slice 8 times and average the values to improve image quality. The resonant scanner will permit fast scanning to avoid bleaching during imaging. If the microscope does not have a resonant scanner, reduce the number of scans to four to avoid bleaching.</li>
</ol>
4. Post Imaging Analysis of Nuclei Number and Distribution
Note: Refer Supplementary Figure 1 for screenshots of the software tools and buttons used.
<ol>
	<li>Import the image to Imaris 8.4.1 or later versions of the software.</li>
	<li>Define mitotic region, transition zone, meiotic region, oocyte region, and spermatheca of the germline by identifying the nuclear morphology in each region.</li>
	<li>Use the surface function button (Supplementary Figure 1A) of the software to select the region of interest.</li>
	<li>Cancel automatic surface creation wizard and manually draw the region of interest at the first and last slice of the image from the three-dimensional stack.</li>
	<li>Click Create surface. 
	NOTE: The software will automatically develop the stacks between first and last stack.</li>
	<li>Mask all channels except DAPI by clicking Mask channel button (Supplementary Figure 1B-C).</li>
	<li>Define nuclear size parameters using the spot function button (Supplementary Figure 1A). For the mitotic region, define an XY diameter of 2 &#181;m while leaving Z diameter undefined. For the transition zone, define an XY diameter of 2 &#181;m and Z diameter as 1.5 &#181;m. (Supplementary Figure 1D). 
	NOTE: Troubleshoot the diameters according to the magnification and specifications of the microscope. For the current protocol, the objective used was 63X and provided an extra 1.70 times magnification during imaging. If the objective changes, for instance 40X, the diameters should be changed accordingly. Troubleshooting should be performed with wildtype germlines to establish the correct parameters. The mitotic region of a wild type germline arm has approximately 250 nuclei. The diameter should be modified to achieve this value. Use at least 15 germlines to determine the optimal value for the diameter. A similar approach should be used for calibrating the transition zone (150 - 170 nuclei) and meiotic region (600 - 700 nuclei).</li>
	<li>Limit the detection of spots by defining the minimum threshold (Supplementary Figure 1E). Use DAPI stained wild type germlines to establish minimum threshold by increasing the minimum three-dimensional rendering threshold until the first spot appears outside the germline.</li>
	<li>Save the image and obtain the number of nuclei from the table generated automatically by the software. Export the images as TIF files.</li>
</ol>
5. Post Imaging Analysis for Scoring Sperm and Chromosome Number
<ol>
	<li>Perform three-dimensional rendering by selecting the spermatheca as the region of interest. To detect each sperm, define the spot diameter between 0.75 - 1.0 &#181;m for X and Y- axis, while Z diameter remains undefined. Use the spots function button as shown in Supplementary Figure 1.</li>
	<li>Subtract the background by ticking background subtract box and using background correction values calculated by the software (Supplementary Figure 1D). 
	NOTE: Imaris software picks high intensity points first, therefore the effect of background is minimal as long as there is significant difference between the region of interest and the background. A second method is to define the background correction manually, where appropriate values can be given for the background. Manual background correction will be appropriate if the intensity of independent samples needs comparison.</li>
	<li>Adjust the three-dimensional rendering threshold to detect all DAPI stained sperm in the spermatheca (Supplementary Figure 1E).</li>
	<li>For chromosome counting, define the spot diameter &#60;0.75 &#181;m for X and Y- axis before developing three-dimensional models.</li>
	<li>Save the image and export as TIF file.</li>
</ol>
6. Post Imaging Analysis for Cytoskeletal Reconstruction of the Germline
<ol>
	<li>Identify the region of interest in the germline and mask all channels except phalloidin by clicking Mask channels button.</li>
	<li>Define a surface detail of 0.25 &#181;m by using the surface function button (Supplementary Figure 1). 
	Note: This means every 0.25 &#181;m will be rendered into three-dimensional models. Surface detail can be constant for any region of the germline where a three-dimensional surface model will be created for every 0.25 &#181;m. However, for the oocyte region, surface detail can be increased to 0.35 - 0.5 &#181;m due the presence of well-defined actin fibers in this region.</li>
	<li>Subtract the background by ticking background subtract box and using background correction values calculated by the software (Supplementary Figure 1D).</li>
	<li>Adjust three-dimensional rendering threshold depending on the structure requiring analysis (Supplementary Figure 1E).</li>
	<li>Save the developed three-dimensional image and export as a TIF file.</li>
</ol>

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The authors declare no conflict of interests.

The goal of this protocol is to improve the accuracy and reduce the time required for germline analysis. After standard preparation of dissected germlines, a three-dimensional model of germline nuclei is prepared by computational rendering. While allowing the observation of germline nuclei distribution in space, three-dimensional rendering calculates the number of nuclei at specific regions of the germline. The critical aspect of our method is accurate definition of size and shape parameters of nuclei. This depends on clarity of staining and magnification used to image. We confirmed the accuracy of the method by comparing to published manually-analyzed data1. To further confirm the accuracy of our method, we used multiple mutants under different conditions and confirmed the results with published literature. It also confirmed the ability of the method to detect subtle and strong phenotypes - cpb-3 and glp-1 mutants, respectively. In addition, this method shows adaptability to changes in nuclei size and shape within the germline or even between strains. This property also enables the identification and scoring of smaller structures in the germline such as chromosomes and sperm. The method also allows further troubleshooting for size and shape parameters, if required. Our analysis was able to elucidate the distribution of nuclei in the germline and showed that nuclei in mitotic and pachytene regions are organized in different patterns. By applying three-dimensional rendering to REC-8 and DAPI staining, we also showed the distribution of REC-8 within nuclei of the mitotic region.
Three-dimensional rendering of actin staining enabled detailed examination of germline cytoskeletal structures. The germline appears to have two separate actin structures, though they maintain physical contact. While the reconstruction of the cytoskeleton for the entire germline is possible as a single entity, the best results can be obtained if the regions of interest are preselected into mitotic, transition, and meiotic regions. The distal germline cytoskeleton has an undefined shape and evolves into a filamentous structure at the proximal end. Our protocol therefore enables three-dimensional reconstruction of the germline actin cytoskeleton by defining specific parameters to accommodate differences in actin distribution. The most critical aspect of the analysis is the definition of the parameters such as nuclei size and surface detail requirement. Lowering the nuclei size below the actual size will risk the software to pick bright spots within the nuclei as separate objects. Similarly, high surface details values will lead to the loss of accurate three-dimensional structures. The proposed method relies on the precision of dissection and the quality of the germline staining. A damaged germline may lead to the release of nuclei from the germline, causing inaccurate numbering. High background from the staining will also cause the inaccurate nuclei count and improper three-dimensional rendering.
Our method extends previously known automated methods of germline analysis where we are able to analyze the nuclei number and distribution/expression of germline proteins in space with a single protocol20. Secondly, our method enables scoring of smaller structures such as chromosomes within the germline. Chromosome segregation is an important aspect of oocyte development21. Using our method, it is possible to detect the number and position of chromosomes where the distance can be defined by calibrating against wild type oocytes. Any mutation affecting the chromosome separation can be studied using this tool. Also, the method can be extended to study chromosomes in embryos at the 2-cell stage. The computational analysis also provides the number of sperm in the spermatheca, number of chromosomes in an oocyte and germline cytoskeletal structures. Taken together, the method provides comprehensive analysis of the C. elegans germline using a single protocol while reducing analysis time considerably. The method eliminates the requirement of multiple tools for each analysis (nuclei number, protein distribution, and cytoskeletal structure) and removes the coding time required in any specific software. Finally, this method can be effectively extended to other worm studies including the three-dimensional analysis of sedated live worms.

The conservation of signaling pathways with mammals makes C. elegans an excellent model to study multiple biological processes1,2. In our lab, we use the C. elegans germline to study stem cell development, apoptosis, and gene expression. While the germline is a three-dimensional structure, many studies are two dimensional due to the time-consuming and labor-intensive nature of three-dimensional analysis. It is highly likely that two-dimensional analysis may misrepresent in vivo events in the germline. The C. elegans adult hermaphrodite has two germline arms, each of which houses a somatic distal tip cell (DTC) that maintains distal germ cells in an undifferentiated state3,4. These germ cells begin to differentiate as they move away from the DTC, escaping its influence, and become oocytes and sperm as they reach the proximal end of the germline. During this process, germ cell nuclei undergo mitosis, before transitioning to meiosis5,6. Sperm production is completed by larval stage 4 (L4) of the development, after which oocytes are produced during the adulthood. The sperm are stored in the spermatheca where they fertilize oocytes to generate embryos.
There are multiple genetic and environmental factors that can influence germline development in C. elegans resulting in changes in the number of nuclei, number of apoptotic events, chromosome dynamics, and protein expression and/or localization7,8,9,10,11. The analysis of these events requires the identification of each stage of differentiation based on nuclear morphology and distribution. To accurately analyze these parameters manually with a large sample size is labor-intensive and time-consuming. To circumvent these drawbacks and to enable the consistency of analysis, we developed an automated method for three-dimensional examination of the C. elegans germline for nuclei counting, nuclei distribution, protein expression, and cytoskeletal structure. By combining confocal microscopy with three-dimensional rendering, we generated size and shape parameters for the identification of each stage of germ cell differentiation. Further, this method enables counting of germ cell nuclei and sperm plus scoring of chromosome number in each oocyte.
One crucial structure in the germline is the cytoskeleton, which provides stability to the germline compartment, aids cytoplasmic streaming and protection to germline nuclei12. Using computational rendering, we performed three-dimensional reconstruction of the germline cytoskeleton and identified distinct cytoskeletal features within the germline. Here, we describe a step-by-step protocol to illustrate how computational analysis combined with confocal imaging enables comprehensive analysis of the C. elegans germline.
We propose a rapid method for the three-dimensional analysis of C. elegans germline (Figure 1). Using three-dimensional analysis, it is possible to study the three-dimensional distribution of germline nuclei (Figure 2 and Figure 3), automated counting of cells (Figure 2), reconstruction of the germline cytoskeleton (Figure 3), distribution of proteins (Figure 4), and scoring the number of sperm in the spermatheca and chromosomes in oocytes (Figure 5). The method not only enables easy and accurate quantification of the germline but identifies physiologically relevant phenotypes.

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="934">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C. elegans strains: wild type (N2, Bristol), rnp-8(tm2435) I/hT2[bli-4(e937) let-?(q782) qIs48] (I;III), cpb-3(bt17) I, glp-1 (e2141) III&#160;</td>
			<td style="width:264px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:168px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OP50 Escherichia coli bacteria</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nematode Growth Media (NGM) plates</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyclonal rabbit anti-REC-8&#160;</td>
			<td>SDIX</td>
			<td>29470002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 conjugated antibody raised in goat</td>
			<td>Thermofisher Scientific</td>
			<td>A-21236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytoskeletal dye phalloidin&#160;</td>
			<td>Thermofisher Scientific</td>
			<td style="width:168px;">A-12380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetramisol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES buffer, pH 7.4&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">G0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X PBS pH 7.4&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P1389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37% Paraformaldehyde solution</td>
			<td>Merck Millipore</td>
			<td>1040031000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroshield fixing reagent&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol&#160;</td>
			<td>Millipore&#160;</td>
			<td>1009832511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20&#176;C &#38; 25&#176;CIncubator&#160;</td>
			<td>Any brand</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope</td>
			<td style="width:264px;">Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;&#160;</td>
			<td style="width:264px;">Any brand (Leica, Zeiss)</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Computer equipped with Imaris suit 8.4.1 or later version, full licence to use the software and Matlab software.</td>
			<td style="width:264px;">Bitplane</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Phospho buffered saline, pH 7.4</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon microscope slides&#160;</td>
			<td style="width:264px;">Tekdon&#160;</td>
			<td style="width:168px;">&#160;941-322-8288</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 indicates the time required for three-dimensional germline analysis. L4 hermaphrodites incubated at 20 &#176;C were dissected to isolate germlines and stained with DAPI, phalloidin, and antibodies against germline proteins. Germlines are imaged using confocal microscopy. Staining and confocal microscopy requires approximately 24 h. Computational analysis for the complete germline requires 10 - 15 min to count the number and position of nuclei, identify the protein distribution, analyze the cytoskeletal structure and score the number of sperm. Complete manual analysis requires over 2 h per germline and is subject to operator influence, therefore, our automated high-throughput method reduces the analysis time and enhances the reproducibility.
Automated nuclei scoring reveals that a germline arm houses approximately 1,000 - 1,200 nuclei (Figure 2C), corresponding well with previously published manual scoring data1. To confirm the accuracy of scoring, multiple mutants and different conditions are used (Figure 2D-G). For instance, we compared rnp-8(tm435), cpb-3(bt17), and glp-1(e2141) mutant worms to wild type animals. Automated counting reproduced the known reduction of number of nuclei in cpb-3 and glp-1 mutant germlines14,15,16,17. A severe reduction in nuclei number was also observed in the glp-1(e2141) temperature-sensitive mutant when incubated at 25 &#176;C. The distribution of nuclei is dependent on the stage of differentiation. The mitotic region, which contains approximately 250 nuclei in wild type, is tightly-packed with nuclei compared to the rest of the germline (Figure 3)1. The spacing between mitotic nuclei is minimal and nuclei are located throughout the mitotic region. However, as the nuclei move away from distal end, they appear more towards the circumference of the germline (Figure 3). The change in distribution starts at the transition zone and completes as the nuclei enter meiosis/pachytene.
The germline has specific cytoskeletal structures at different stages of differentiation. We visualized F-actin by staining the germline with phalloidin. The distribution of F-actin from the distal to proximal end of the germline was distinct at each region (Figure 3 and Figure 5). In general, there are two separate structures that appear as a &#39;cylinder within cylinder.&#39;&#160;At the mitotic region, inner actin appears as a solid mass with a specific shape (Figure 3). As the germline reaches the transition zone, the inner actin assumes a more cylindrical structure and it becomes a hollow cylinder as it reaches late pachytene. The second layer of actin covers the inner layer giving shape to the germline. This &#39;cylinder within cylinder&#39; actin structure disappears towards the oocyte region (Figure 5). At the oocyte region, actin appears as thick fibers. The oocytes are separated by an actin rachis, though it appears to be dynamic to allow the movement of oocytes to the spermatheca. Finally, actin in the spermatheca also forms thick bundles of actin fibers. However, the fibers appear to be packed closer than in the oocyte region. The cytoskeletal structure appears to complement the distribution patterns of nuclei (Figure 3). At the mitotic region, the nuclei appear to be placed around the inner actin structure. As they reach meiosis/pachytene, the nuclei organize between the two actin cylinders leaving the middle of the germline mostly devoid of nuclei (Figure 3). This could probably aid uninterrupted cytoplasmic streaming in the germline. Germlines were stained with an antibody against the REC-8 protein and analyzed by confocal microscopy. REC-8 is homogeneously distributed around early nuclei of the germline and is later cleaved and degraded during meiosis18. REC-8 distribution in three-dimensional cross-sectional analysis of the germline shows the distribution of protein in the mitotic region (Figure 4).
Three-dimensional rendering of the proximal end of the germline includes the spermatheca and first three oocytes distal to the spermatheca. Our analysis recognized an average of 151 sperm in each spermatheca (n = 18). This corresponds to the published literature, indicating the reliability of the method (Figure 5F)19. This analysis may be performed together with cytoskeletal or protein distribution studies. The C. elegans genome is distributed among six chromosomes. In fully developed wild type oocytes, these chromosomes are visible and can be counted. This chromosome separation can be used to study chromosome stability or other defects associated with oocyte development. By three-dimensional rendering, the number of chromosomes can be visualized, and if the chromosomes are separated properly, the number can be accurately obtained (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57702/57702fig1.jpg" /> 
Figure 1. Timeline for automated analysis. Time comparison between manual and computational analysis of the C. elegans germline. <a href="/files/ftp_upload/57702/57702fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57702/57702fig2.jpg" /> 
Figure 2. Nuclei distribution in the germline. (A) DAPI stained germline showing mitotic, transition, and pachytene/meiotic regions. (B) Computational three-dimensional model of the germline. (C) Scoring for the total number of nuclei in wild type germlines. (D-F) Scoring for the number of nuclei at mitotic, transition, and meiotic regions of wild type, rnp-8, cpb-3, and glp-1 mutant germlines. (G) Comparison in the total number of nuclei in the glp-1 mutant at 20 &#176;C and 25 &#176;C. Error bar represents standard error. Student&#39;s test. ****n&#160;&#60;0.0001, ***n&#160;&#60;0.001, **n&#160;&#60;0.01, *n&#160;&#60;0.05. <a href="/files/ftp_upload/57702/57702fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57702/57702fig3.jpg" /> 
Figure 3. Cytoskeletal structure of the germline. (A-B) Organization of the inner actin (red) cytoskeleton of the germline at the mitotic region. The inner actin has a diffuse structure compared to outer actin at the mitotic region. (C-D) At the pachytene region, the cytoskeleton assumes a cylindrical structure by forming two cylinders between which the nuclei (blue) are organized. (E-F) Organization of germline nuclei in the mitotic and pachytene regions. The distribution of nuclei is more towards the circumference of the &#39;germtube&#39; at the pachytene region compared to mitotic region (green) and middle of the germline become devoid of nuclei. <a href="/files/ftp_upload/57702/57702fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57702/57702fig4.jpg" /> 
Figure 4. REC-8 expression in the mitotic region. (A) Micrograph showing the distal end of a REC-8-stained germline. (B-C) Three-dimensional rendering of REC-8 staining (shown in blue) and distribution of protein between the mitotic nuclei (green). REC-8 staining is less abundant proximal to the mitotic region (marked by white dots representing nuclei). <a href="/files/ftp_upload/57702/57702fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57702/57702fig5.jpg" /> 
Figure 5. Germline structure at the proximal end. (A) DAPI staining of the germline at the proximal end. (B) Three-dimensional rendering of DAPI staining showing sperm in the spermatheca (yellow) and chromosomes in the oocytes (white). (C-E) Three-dimensional cytoskeletal structure at the proximal end of the germline. Red marks the complete proximal end and green marks the spermatheca. Cross sectional analysis shows that the cytoskeleton not only forms a filamentous structure of actin around oocytes, but also separates between oocytes. (F) Graph showing the number of sperm in each spermatheca and average obtained from multiple spermatheca (n = 18). Error bar represents standard error. <a href="/files/ftp_upload/57702/57702fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/57702/57702supfig1.jpg" /> 
Supplementary Figure 1. Software tools description. (A) The spots and surface tools for detecting nuclei and protein staining. (B-C) The selection of three-dimensional region of interest using the surface tool. (D) Defining the XY-diameter, using spots tool, for nuclei detection. Depending on the region of interest and staining, the Z-axis diameter and background correction can be used. (E) Minimum and maximum intensity threshold detection. A similar approach can be used for surface function where instead of defining the diameter, and the surface details can be defined. <a href="/files/ftp_upload/57702/57702supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

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.

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 ...

computational-analysis-caenorhabditis-elegans-germline-to-study

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.2K Views.  Monash University. 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.

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

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 ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with 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 ...

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.

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.

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

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 ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

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 ...

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 ...

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 ...