In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Summary

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

Abstract

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

Introduction

Phenotypic plasticity, including developmental and behavioral plasticity, is important for adaptations to adverse environmental conditions and is considered a driving force in evolution¹. C. elegans is a model organism frequently used for studies in development and neurobiology. Under ideal conditions, C. elegans develops through four larval stages prior to entering the adult reproductive stage. However, under conditions of low food availability and high population densities, C. elegans can undergo a developmental switch and enter into a long-lived and stress-resistant dauer stage². Dauers display several morphological ....

Protocol

1. Crude Dauer Pheromone Production

1. Grow OP50 E. coli from a single colony O/N at 37 °C while shaking in 100 ml of LB broth at 200 rpm. NOTE: This O/N culture can be made in advance and stored at 4 °C.
2. Grow N2 C. elegans on 15 - 20 6 cm Petri dishes with NGM agar (See recipe in Table 1) seeded with 50 µl of OP50 E. coli until the bacteria is almost depleted¹⁹.
3. Make 5x 250 ml of S media (See recipe in Table 1) in 1 L Erlenmeyer flasks¹⁹. NOTE: This solution can be made in advance.
4. Grow a culture of OP50 E. coli in 4 L of LB broth O/N by inoculating with 1 ....

Results

The production of crude dauer pheromone results in a yellow liquid (Figure 1) that is both heat and cold stable. Aliquots of crude pheromone are stored at -20 °C indefinitely with no obvious loss in activity. Following the production of pheromone, a single dose-response bioassay is sufficient for a rough estimate of potency. However, for most experiments it is necessary to repeat the bioassay 2 - 3 times and take an average from each experiment. Representative results from two pheromone bioassays are giv.......

Discussion

An examination of dauer-specific neuroplasticity and other dauer-associated morphological changes requires controlled and reliable formation of dauers either through genetic mutation (i.e., daf-c mutants) or by exposure of wild-type animals to pheromone. While the use of a genetic mutation to induce dauers is convenient, it may confound results. Therefore data collected with daf-c mutations should be subsequently confirmed with wild-type animals induced to enter the dauer stage by exposure to d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N2 C. elegans Bristol strain	Caenorhabditis Genetics Center
PT2660 myIs13[Pklp-6::gfp] III	Upon request from Schroeder lab
PT2762 myIs14[Pklp-6::gfp] V	Upon request from Schroeder lab
JK2868 qIs56[Plag-2::gfp] V	Caenorhabditis Genetics Center
OP50 E. coli	Caenorhabditis Genetics Center
Petri dishes	Tritech Research	60 mm T3308, 35 mm T3501
NGM agar	Various		3 g NaCl, 17 g agar, 2.5 g peptone in 975 ml H2O autoclaved cooled to 55°C followed by 1 ml of 5 mg/ml cholesterol in ethanol, 25 ml of 1 M KPO4 buffer (pH 6.0), 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4
S Media	Various		S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 ml. Autoclaved.) To the S basal solution add 2.5 ml of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 ml, autoclaved), 2.5 ml of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4 •7 H2O, 0.2 g MnCl2•4 H2O, 0.29 g ZnSO4 •7 H2O, 0.025 g CuSO4 •5 H2O, H2O to 1 L. Autoclaved and stored in the dark), 0.75 ml of 1 M CaCl2, and 0.75 ml of 1 M MgSO4.
Dauer pheromone plates for dose-response assay	Various		Mix 0.085 g Noble agar, 0.5 ml of 0.513 M NaCl and 5 µl of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 ml culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 ml.  Boil over a Bunsen burner until the agar is dissolved and then cool to 55 °C. Add 125 µl 1 M KPO4 buffer (pH 6.0), 50 µl 0.1 M CaCl2, 50 µl 0.1 M MgSO4 and 50 µl of 5 mg/ml streptomycin sulfate to each tube.  Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).
Microsphere beads 0.1 micron	Polysciences Inc.	00876-15
M9 buffer	Various		Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving