title: A high-content assay for identifying small molecules that reprogram C. elegans germ cell fate.
abstract: Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Benson
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Cummings
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA.
authors.forename: Linda P
authors.initials: LP
authors.lastname: O'Reilly
authors.affiliation: Department of Oncology, Department of Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA; Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: leemy@ecu.edu.
authors.forename: Myon-Hee
authors.initials: MH
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of UPMC, 4401 Penn Avenue, Pittsburgh, PA 15224, USA. Electronic address: paksc@upmc.edu.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pak
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000254:growth & development; D019070:Cell Lineage / Q000187:drug effects; D065150:Cellular Reprogramming / Q000187:drug effects*; D005854:Germ Cells / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D008297:Male; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000008:administration & dosage*
chemical_list: D054852:Small Molecule Libraries
keywords: C. elegans; Cell fate; Drug screening; High-content screening; Reprogramming