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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

The conservation of signaling pathways with mammals makes C. elegans an excellent model to study multiple biological processes^1,2. 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 state^3,4. 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 meiosis^5,6. 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 localization^7,8,9,10,11. 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.

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Protokół

1. Preparation and Worm Husbandry

Note: Refer Table of Materials for all product information.

1. 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 °C without antibiotics.
2. Seed 400 µL 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.
3. Pick worms on seeded NGM plates and incubate at 15 °C or 20 °C.
   1. For the analysis of the adult hermaphrodite germline, pick well-fed L4 worms to the bacterial lawn and incubate overnight at 20 °C.
   2. For scoring sperm number in hermaphrodites, incubate L4 worms at 20 °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.
4. Prepare Teflon microscope slides by placing 25 µL 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 °C for 15 - 20 min.

2. Germline Dissection and Staining^6,13

1. Spot 10 µL of 0.01% tetramisole (anesthetic) on a 22 mm × 22 mm coverslip and pick one-day old adult worms into it.
2. Dissect the worm at the tail as it becomes sedated using a syringe (5 mL) and a needle (24" x 1"), 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.
3. Place the inverted coverslip on a poly-L-lysine coated slide, keeping the dissected germline on the slide.
4. 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.
5. Transfer the slides into -20 °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',N'-tetraacetic acid (EGTA), and 3.7% paraformaldehyde) at room temperature.
6. 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.
7. Block the germlines with 30 µL of blocking solution (30% of goat serum prepared by adding 900 µL of goat serum in 1700 µL of distilled H₂O and 300 µL of 10x PBS) in a humid chamber prepared by placing wet paper towel in a plastic box at room temperature for 30 min.
8. Add 50 µL of REC-8 antibody solution prepared in 30% goat serum in a ratio 1:300 to each sample. Incubate overnight at 4°C.
9. 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.
10. 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.
11. Add 50 µL of secondary antibody solution to the slide.
12. Incubate for 1 h at room temperature.
13. Wash the slides twice by placing in a chamber containing 1x PBS with 0.1% Tween-20 for 10 min. Wipe off excess liquid.
14. Add 30 µL of fixing reagent onto the slide and place a 12 mm² x 1.5 µm coverslip on top and dry the slides at 4 °C overnight.

3. Confocal Microscopy

1. 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.
2. 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.
3. Image the entire germline by defining the thickness of each slice up to 0.5 µm. 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.

4. Post Imaging Analysis of Nuclei Number and Distribution

Note: Refer Supplementary Figure 1 for screenshots of the software tools and buttons used.

1. Import the image to Imaris 8.4.1 or later versions of the software.
2. Define mitotic region, transition zone, meiotic region, oocyte region, and spermatheca of the germline by identifying the nuclear morphology in each region.
3. Use the surface function button (Supplementary Figure 1A) of the software to select the region of interest.
4. 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.
5. Click Create surface.
   NOTE: The software will automatically develop the stacks between first and last stack.
6. Mask all channels except DAPI by clicking Mask channel button (Supplementary Figure 1B-C).
7. Define nuclear size parameters using the spot function button (Supplementary Figure 1A). For the mitotic region, define an XY diameter of 2 µm while leaving Z diameter undefined. For the transition zone, define an XY diameter of 2 µm and Z diameter as 1.5 µm. (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).
8. 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.
9. Save the image and obtain the number of nuclei from the table generated automatically by the software. Export the images as TIF files.

5. Post Imaging Analysis for Scoring Sperm and Chromosome Number

1. 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 µm for X and Y- axis, while Z diameter remains undefined. Use the spots function button as shown in Supplementary Figure 1.
2. 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.
3. Adjust the three-dimensional rendering threshold to detect all DAPI stained sperm in the spermatheca (Supplementary Figure 1E).
4. For chromosome counting, define the spot diameter <0.75 µm for X and Y- axis before developing three-dimensional models.
5. Save the image and export as TIF file.

6. Post Imaging Analysis for Cytoskeletal Reconstruction of the Germline

1. Identify the region of interest in the germline and mask all channels except phalloidin by clicking Mask channels button.
2. Define a surface detail of 0.25 µm by using the surface function button (Supplementary Figure 1).
   Note: This means every 0.25 µm 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 µm. However, for the oocyte region, surface detail can be increased to 0.35 - 0.5 µm due the presence of well-defined actin fibers in this region.
3. Subtract the background by ticking background subtract box and using background correction values calculated by the software (Supplementary Figure 1D).
4. Adjust three-dimensional rendering threshold depending on the structure requiring analysis (Supplementary Figure 1E).
5. Save the developed three-dimensional image and export as a TIF file.

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Wyniki

Figure 1 indicates the time required for three-dimensional germline analysis. L4 hermaphrodites incubated at 20 °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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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
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	Caenorhabditis Genetics Center (CGC)
OP50 Escherichia coli bacteria	Homemade
Nematode Growth Media (NGM) plates	Homemade
polyclonal rabbit anti-REC-8	SDIX	29470002
Alexa 488 conjugated antibody raised in goat	Thermofisher Scientific	A-21236
Cytoskeletal dye phalloidin	Thermofisher Scientific	A-12380
DAPI	Thermofisher Scientific	62248
Poly-L-lysine	Sigma Aldrich	P5899
Tetramisol	Sigma Aldrich	P5899
MgSO4	Sigma Aldrich	M7506
1M HEPES buffer, pH 7.4	Sigma Aldrich	G0887
10X PBS pH 7.4	Thermofisher Scientific	AM9625
Tween-20	Sigma Aldrich	P1389
EGTA	Sigma Aldrich	E3889
37% Paraformaldehyde solution	Merck Millipore	1040031000
Normal goat serum	Sigma Aldrich	G9023
Fluoroshield fixing reagent	Sigma Aldrich	F6182
Ethanol	Millipore	1009832511
Methanol	Sigma Aldrich	34860
20°C & 25°CIncubator	Any brand
Light microscope	Any brand
Confocal microscope	Any brand (Leica, Zeiss)
Computer equipped with Imaris suit 8.4.1 or later version, full licence to use the software and Matlab software.	Bitplane
Phospho buffered saline, pH 7.4	Homemade
Teflon microscope slides	Tekdon	941-322-8288