Format

Send to

Choose Destination

See 1 citation:

PLoS One. 2015 Jul 10;10(7):e0131908. doi: 10.1371/journal.pone.0131908. eCollection 2015.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

Author information

1
Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
2
Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
3
Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.

Abstract

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

PMID:
26161877
PMCID:
PMC4498767
DOI:
10.1371/journal.pone.0131908
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center