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Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans
Published on: February 22, 2015
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A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate
Joshua A Benson1, Erin E Cummings1, Linda P O'Reilly1
1Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
Methods (San Diego, Calif.)
|July 4, 2014
Summary
Researchers developed a high-throughput workflow to screen compounds that reprogram cell fates in C. elegans. This method uses a novel assay to identify chemical modulators of germ cell reprogramming, restoring fertility in sterile mutants.
Area of Science:
- Developmental Biology
- Genetics
- Pharmacology
Background:
- Committed cell fates can be reprogrammed through genetic, chemical, and environmental factors.
- The nematode Caenorhabditis elegans offers a model organism for studying cell fate reprogramming in vivo.
- Previous work identified puf-8; lip-1 mutants with enhanced MPK-1/MAPK signaling, leading to sterility.
Purpose of the Study:
- To establish a high-throughput screening (HTS) workflow for identifying compounds that modulate germ cell fate reprogramming in C. elegans.
- To overcome challenges associated with using puf-8; lip-1 mutants in HTS, such as their sterility and the need for specialized sorting and assaying.
- To develop a robust assay for measuring the effects of compounds on germ cell reprogramming and restoring fertility.
Main Methods:
- Utilized the COPAS™ BIOSORT for semi-automated sorting of homozygous puf-8; lip-1 mutants into 384-well plates.
- Developed an image-based assay to quantify germ cell reprogramming by measuring viable progeny.
- Employed puf-8; lip-1 mutants with enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling.
Main Results:
- Successfully established a semi-automated workflow for HTS of germ cell fate modulators.
- Developed a high-content, image-based assay to accurately measure compound efficacy.
- Demonstrated the potential of this workflow to identify compounds that restore fertility by reprogramming germ cell fate.
Conclusions:
- The described workflow enables high-throughput screening for chemical modulators of germ cell reprogramming in C. elegans.
- This approach facilitates the study of cell fate plasticity within a whole-organism context.
- The developed methods are crucial for advancing the discovery of compounds that influence developmental processes.
