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

Summary

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.

Abstract

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.

Introduction

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 ho....

Protocol

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 ....

Results

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, identify th.......

Discussion

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.......

Materials

List of materials used in this article

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