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 cl...

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

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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

Universidad de Cartagena UDEC

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

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 ...

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

Genetics

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 ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

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

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

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

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

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

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biology

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Neuroscience

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

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

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Chapters in this video

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

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

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

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

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

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans