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

Summary

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.

Abstract

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.

Introduction

With the steady improvements of fluorescent proteins, genome engineering, light microscopy, and computer soft- and hardware, it is now possible to record development of many model organisms at unprecedented spatio-temporal resolution. This allows researchers to ask questions that could not be addressed previously or to revisit known developmental processes in order to search for overlooked aspects. This progress has sparked the field of quantitative developmental biology, which aims at transforming qualitative, informal models into quantitative models by thorough measurements and statistical analyses.

Tracking cells and subcellular structur....

Protocol

1. Preparation of C. elegans Embryos for Time-lapse Microscopy using Microbeads

1. Transfer gravid adult worms by using a worm pick (platinum wire) into a drop of M9 buffer and clean off bacteria by first vigorously agitating and then transferring each worm to an adjacent drop of M9 using an eye lash mounted onto a tooth pick/pipette tip or use a pointed glass capillary.
2. Dilute the dispersion containing the 20 µm diameter polystyrene beads 10-fold in M9 buffer (1 L M9 buffer contains 3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, and, after autoclaving (20 min, 120 °C), add an additional 1 ml of a 1....

Results

By using protocols 2, 3, and 4, time-lapse imaging of gonads in wild type C. elegans adults is performed (strain OD58 (unc-119(ed3) III; ltIs38[pAA1; pie-1::GFP::PH(PLC1delta1) + unc-119(+)]), expressing a membrane marker from a germline promoter). Focusing on the turn of the gonad, a 3D model of the germ cells is generated from the microscopy data (Figure 2). This model allows to analyze changes in cell size while the cells transit form the distal to the proximal arm, reveals the organization o.......

Discussion

Through object tracking in development, in particular nuclear tracking, it has been possible to elucidate central patterning mechanisms of C. elegans embryogenesis^1,23,24. Expanding this strategy to higher throughput, it has been recently possible to uncover additional patterning rules and to propose a method how to deduce patterning rules de novo¹⁰. For many mutants, however, the precise patterning defects are still unknown. The methods described here are tools that can be instrum.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereo microscope	Motic/VWR	OT4005S	Stereo microscope for dissection and mounting
Polybead Polystyrene Microspheres,	Polysciences	18329	Embryo mounting
20 µm
Polybead Polystyrene Microspheres,	Polysciences	876	Adult animal mounting
0.1 µm
Microscope slides	VWR	631-0902	Adult animal mounting
Cover glass 18x18 mm	VWR	631-1331	Embryo/adult mounting
Cover glass 24x60 mm	VWR	631-1339	Embryo mounting
Scalpel	VWR	233-5455	Embryo dissection
Silicone tubing	VWR	228-1501	Tubing for mouth pipette
30 mm PTFE membrane filter	NeoLab	Jul-01	Filter for mouth pipette
Capillary tubes	VWR	621-0003	Pipette tip for mouth pipette
Vaseline	Roth	E746.1	Embryo/adult mounting
Agar	Roth	5210.5	Adult animal mounting
Potassium-di-hydrogenphosphate	Roth	P018.2	M9 buffer
Di-sodium- hydrogenphosphate	Roth	P030.2	M9 buffer
Sodium chloride	Roth	3957.1	M9 buffer
VisiScope Spinning Disc Confocal System	Visitron Systems	n/a	Confocal microscopy