Name: tracking and quantifying developmental processes in c. elegans using open-source tools
Uploaded: 2015-12-16T13:00:00
Description: Watch this Scientific Journal Video about Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools at JoVE.com

The authors have nothing to disclose.

1. Preparation of C. elegans Embryos for Time-lapse Microscopy using Microbeads
<ol>
	<li>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.</li>
	<li>Dilute the dispersion containing the 20 &#181;m diameter polystyrene beads 10-fold in M9 buffer (1 L M9 buffer contains 3 g ...

<ol>
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Through object tracking in development, in particular nuclear tracking, it has been possible to elucidate central patterning mechanisms of C. elegans embryogenesis1,23,24. 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...

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 structures has made it possible to derive quantitative models of embryonic development, nervous system activity, or cell division1-12. By tracking cell division remnants during early development of the C. elegans embryo, we could recently reveal that they follow a stereotyped path and constitute important polarizing factors13,14.
Here, protocols are presented that make quantitative developmental biology approaches accessible for non-experts. The focus lies on three straight-forward, freely available tools that are implementable in any lab that has access to standard confocal microscopy and computers. These include a protocol to generate 3D cell shapes, a protocol to track cell division remnants, and a protocol to quantitatively describe cortical actomyosin dynamics. The nematode C. elegans is used as an exemplary case, however, the methods and tools discussed here are suited for a variety of questions in other biological models, e.g., cultured cells, tissue explants, organoids or spheroids, other embryos, etc.
Generally, some of the analyses shown here can also be performed with the popular open source tool ImageJ (http://imagej.nih.gov/ij/docs/index.html; or FIJI, the &#8216;batteries included&#8217; version of ImageJ, http://fiji.sc/Fiji) for which many plugins for different quantitative analyses are available. However, the programs discussed here are designed to tackle specific problems.
Firstly, IMOD, an image processing, modeling and display suite can be used for 3D reconstructions of serial sections from electron or light microscopy15. IMOD also contains tools for viewing the 3D data from any orientation. Secondly, Endrov, a Java program designed to perform image analysis, data processing, and annotation of networks or tracks (among others) on the basis of an extended plugin architecture, with ImageJ plugin compatibility16. It contains over 140 image-processing operations and an extensible user interface in which model and raw data are displayed separately. Its source code can be found at https://github.com/mahogny/Endrov. Thirdly, PIVlab, a MATLAB tool for digital particle image velocimetry that allows the user to quantitatively and qualitatively analyze particle flow fields17. The use of this programs requires a MATLAB license that includes the Image Processing Toolbox (http://mathworks.com). PIVlab is a program designed to quantitatively describe flow. It calculates the velocity distribution, magnitude, vorticity, divergence, or shear within image pairs or series. For this, it cross-correlates small areas of images (called &#8216;passes&#8217; in the protocol section) of an image pair to derive the most probable particle displacement. This cross-correlation yields a correlation matrix that can be analyzed in the space or frequency domain using either direct cross-correlation or a fast Fourier transformation (FFT), respectively.
The equipment used here is an inverted microscope equipped with a Nipkow (&#39;spinning&#39;) disc, an EMCCD, 488 and 561 nm standard solid-state lasers, and 20x air or 40x or 60x plan/apochromat oil- or water-immersion objectives. However, it is also possible to perform time-lapse imaging with other imaging modalities, e.g., point-, line- or sheet-scanning laser-based microscopy, multi-photon microscopy, as well as epi-fluorescence microscopy combined with deconvolution or structured illumination. The advantage of using a Nipkow disc system is the extremely fast image acquisition, especially if a streaming mode (continuous movement and scanning of the object in the z dimension) is available. In addition, to improve resolution, a 1.5-fold magnifying extender in front of the EMCCD can be used.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">Motic/VWR</td>
			<td style="width:119px;">OT4005S</td>
			<td style="width:167px;">Stereo microscope for dissection and mounting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polybead Polystyrene Microspheres,&#160;</td>
			<td rowspan="2" style="width:71px;">Polysciences</td>
			<td rowspan="2" style="width:119px;">18329</td>
			<td rowspan="2" style="width:167px;">Embryo mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">20 &#181;m</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polybead Polystyrene Microspheres,&#160;</td>
			<td rowspan="2" style="width:71px;">Polysciences</td>
			<td rowspan="2" style="width:119px;">876</td>
			<td rowspan="2" style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">0.1 &#181;m</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microscope slides</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-0902</td>
			<td style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cover glass 18x18 mm</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-1331</td>
			<td style="width:167px;">Embryo/adult mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cover glass 24x60 mm</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-1339</td>
			<td style="width:167px;">Embryo mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Scalpel</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">233-5455</td>
			<td style="width:167px;">Embryo dissection</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Silicone tubing</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">228-1501</td>
			<td style="width:167px;">Tubing for mouth pipette</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">30 mm PTFE membrane filter</td>
			<td style="width:71px;">NeoLab</td>
			<td style="width:119px;">Jul-01</td>
			<td style="width:167px;">Filter for mouth pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Capillary tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">621-0003</td>
			<td style="width:167px;">Pipette tip for mouth pipette</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vaseline</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">E746.1</td>
			<td style="width:167px;">Embryo/adult mounting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Agar</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">5210.5</td>
			<td style="width:167px;">Adult animal mounting</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Potassium-di-hydrogenphosphate</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">P018.2</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Di-sodium- hydrogenphosphate</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">P030.2</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium chloride</td>
			<td style="width:71px;">Roth</td>
			<td style="width:119px;">3957.1</td>
			<td style="width:167px;">M9 buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">VisiScope Spinning Disc Confocal System</td>
			<td style="width:71px;">Visitron Systems</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">Confocal microscopy</td>
		</tr>
	</tbody>
</table>

tracking and quantifying developmental processes in c. elegans using open-source tools

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.

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...

Watch this Scientific Journal Video about Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools at JoVE.com

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

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.

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

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

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Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

Antibody Staining in C. Elegans Using "Freeze-Cracking"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...