Name: computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton
Uploaded: 2018-04-19T12:30:00
Description: 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

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...

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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 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, identi...

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 ...

The overall goal of this method is to automate the analysis of a Caenorhabditis elegans germline to study its nuclei, protein, and cytoskeletal distribution. This method can help answer the key questions in the C.Elegans field about the number of nuclei and sperm, nuclear distribution, and cytoskeletal structure of the germline. The main advantages of this technique are it reduces the analysis time, increases the sample size, and eliminate the human error associated with the manual analysis.To image the samples, place slides with stained germlines into the slide holder above the 63X objective of a confocal microscope, and locate a germline. Focus on the top of the germline, and mark it with the confocal microscope software. When the total germline thickness has been established, define the thickness of each slice up to 0.5 micrometers, and mark the complete germline.Then, start the acquisition, scanning each slice eight times and averaging the values to improve the image quality. When all of the images have been obtained, import the images to an appropriate image analysis software program, and use the surface function tool to select the mitotic region. An image of the distal end of the germline will appear.Cancel the automatic surface creation wizard, and manually draw a region of interest around the mitotic region at the first and last slices of the image from the three-dimensional stack. Click Create Surface, followed by Mask Channel, to mask all of the channels except the DAPI channel. Using the spot function tool, define the size parameters of the mitotic region with an XY diameter of two micrometers and with the Z diameter undefined.Click subtract background to subtract the background. To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files.For the transition zone, create surface as explained before. Then, click Mask Channel to mask all the channels except the DAPI channel. Using spot function, define an XY diameter of two micrometers and Z diameter of 1.5 micrometers, and click subtract background to subtract the background.To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files. For scoring the sperm number, select the spermatheca as the region of interest, as demonstrated for the nuclei regions, and use the spot function tool to define the XY diameter between 0.75 and one micrometers with an undefined Z diameter to detect each sperm.Select the Background Subtract box, and use the background correction values calculated by the software to subtract the background. Then, adjust the 3D rendering threshold to detect all of the DAPI-stained sperm in the spermatheca. For chromosome counting in the oocytes, select the oocytes and define the XY diameter as less than 0.75 micrometers, and define the threshold before developing the 3D models.Then, save the images for export as TIFF files. For cytoskeletal germline reconstruction, identify the mitotic region of interest in the germline and create a surface mask as previously demonstrated. Click Mask Channels to mask all of the channels except the phalloidin channel, and use the surface function tool to define a surface detail of 0.25 micrometers.After subtracting the background, adjust the 3D rendering threshold to save the developed 3D image for export as a TIFF file. The nuclei distribution is dependent on the stage of differentiation. For example, at the mitotic region, the inner actin appears as a solid mass upon which the nuclei are organized.In a wild-type germline, the mitotic region contains approximately 250 nuclei and is tightly packed with nuclei compared to the rest of the germline. As the nuclei move away from the distal end, however, they appear closer to the circumference of the germline, with the change in distribution starting at the transition zone and finishing as the nuclei enter the third stage of prophase in meiosis. At the transition zone, the germline inner actin assumes a more cylindrical structure, becoming a hollow cylinder as it reaches the pachytene.The second layer of actin covers the inner layer, giving shape to the germline. This cylinder within a cylinder actin structure disappears toward the oocyte region, at which point the actin appears as thick fibers covering the oocytes. In the spermatheca, the actin also forms into thick bundles of fiber, although these fibers appear to be packed more closely together than in the oocyte region.Three-dimensional rendering of the proximal end of the germline, as demonstrated, reveals an average of 151 sperm in each spermatheca. Once mastered, this analysis can be completed in 10 minutes per germline. While attempting this procedure, it is important to remember to define the analysis parameters according to the magnification of the objective used to visualize the germline in your experiment.After watching this video, you should have a good understanding of how to perform an automatic analysis of a germline and to study cytoskeletal structure and to score the number of the nuclei within the germline.

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Research

JoVE Journal

Developmental Biology

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 ...

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 ...

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

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

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

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

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