A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

Summary

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Abstract

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80–100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

Introduction

The C. elegans embryo is an important model system for mechanistic cell biology and analysis of cell fate specification and morphogenetic events driving embryonic development^{1,2,3,4,5,6,7,8,9}. To date, much of the characterization of both cellular-level events and cell fate specification in the embryo has been achieved using relatively high temporal resolution ....

Protocol

1. Preparing C. elegans Embryos for Semi-high-throughput Imaging

NOTE: The goal of this portion of the protocol is to load a population of semi-synchronized (2 to 8-cell stage) C. elegans embryos, dissected from suitable marker strains (Figure 2), into a glass-bottom 384-well plate for imaging. Other plate formats could also work, but the 384 well plates are preferred because the small well size constrains the spread of embryos to a relatively small area, which facilitates the identification of fields containing multiple embryos for time-lapse imaging. Roughly synchronizing the ....

Results

A significant challenge in characterizing the effect of molecular perturbations on C. elegans embryonic development is that it takes about 10 h for embryos to progress from first cleavage to the end of elongation at 20°¹⁶. A semi-high-throughput method in which large cohorts of embryos can be simultaneously imaged is useful for events on this time-scale because it permits imaging of multiple conditions in parallel with a sufficient ensemble size for each condition to enable quantitat.......

Discussion

This work describes a suite of tools and methods that were developed to enable larger-scale efforts to profile the function of genes in embryonic development in C. elegans. Our semi-high-throughput method allows 3D time-lapse imaging of embryonic development at 20 min resolution for 80–100 embryos in a single experiment. While this protocol can be adapted for use with any desired marker strain(s), this work demonstrates the potential of the method using two custom strains developed to monitor events during.......

Materials

Name	Company	Catalog Number	Comments
Aspirator Tube Assembly	Drummond Scientific	2-000-000
Calibrated Pipette (25mL)	Drummond Scientific	2-000-025
Cell Voyager Software	Yokogawa Electric Corp	Included with CV1000
Conical Tube (15 mL )	USA Scientific	1475-0501
CV1000 Microscope	Yokogawa Electric Corp	CV1000
Depression slide (3-well)	Erie Scientific	1520-006
Dissection Microscope	Nikon	SMZ-645
Eppendorf Centrifuge 5810R	Eppendorf	5811 07336
ImageJ/FIJI	Open Source	https://imagej.net/Fiji
M9 Buffer	Lab Prepared	https://openwetware.org/wiki/M9_salts
Microcentrifuge Tube (1.5 mLl)	USA Scientific	1615-5500
Microseal F-foil Seal	Bio-Rad	MSF1001
NGM Plates	Lab Prepared	http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html#d0e214
Scalpel #15	Bard Parker	REF 371615
Sensoplate Plus, 384 Well, F-bottom, Glass Bottom	Greiner Bio-One	781855
Tetramisole Hydrochloride	Sigma Aldirch	T1512-10G
Tweezers, Dumont #3	Electron Microscopy Sciences	0109-3-PO
U-PlanApo objective (10× 0.4NA)	Olympus	1-U2B823
U-PlanApo objective (60× 1.35 NA)	Olympus	1-U2B832