title: Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.
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.
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Christoph
authors.initials: C
authors.lastname: Seiler
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Nichole
authors.initials: N
authors.lastname: Gebhart
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Shinton
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Yue-sheng
authors.initials: YS
authors.lastname: Li
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Nicola L
authors.initials: NL
authors.lastname: Ross
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Xingjun
authors.initials: X
authors.lastname: Liu
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Qin
authors.initials: Q
authors.lastname: Li
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Alison N
authors.initials: AN
authors.lastname: Bilbee
authors.affiliation: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Gaurav K
authors.initials: GK
authors.lastname: Varshney
authors.affiliation: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: LaFave
authors.affiliation: Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Burgess
authors.affiliation: Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Jorune
authors.initials: J
authors.lastname: Balciuniene
authors.affiliation: Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Darius
authors.initials: D
authors.lastname: Balciunas
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Hardy
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Dietmar J
authors.initials: DJ
authors.lastname: Kappes
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: David L
authors.initials: DL
authors.lastname: Wiest
authors.affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rhodes
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006410:Hematopoiesis; D016296:Mutagenesis; D013601:T-Lymphocytes / Q000502:physiology*; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D029961:Zebrafish Proteins; C000606720:epidermal growth factor receptor pathway substrate 15-like 1, zebrafish; C000606719:ATP-GTP binding protein 1, zebrafish; D002268:Carboxypeptidases