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

Seiler C, Gebhart N, Zhang Y, Shinton SA, Li YS, Ross NL, Liu X, Li Q, Bilbee AN, Varshney GK, LaFave MC, Burgess SM, Balciuniene J, Balciunas D, Hardy RR, Kappes DJ, Wiest DL, Rhodes J - PLoS ONE (2015)

Bottom Line: An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults.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.

View Article: PubMed Central - PubMed

Affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, 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.

No MeSH data available.


Related in: MedlinePlus

Endogenous gene expression matches the gene-trap GFP expression pattern for agtpbp1 and eps15L1 lines.(A) The GFP pattern in a representative 6 dpf embryo from the agtpbp1fcc301 line. (B-D) WISH of agtpbp1 at 6 dpf. Lateral line cells (*), epiphysis (arrowhead) and nasal pit (arrow) are indicated. (C-D) Magnified views of regions of the embryo shown in B. (E) The GFP pattern in a representative 6 dpf embryo from line eps15L1fcc436. (F-I) WISH of eps15L1 at 6 dpf. Pancreas (under *), kidney (arrowhead) and skin cells (arrows) are indicated. (G-I) Magnified views of regions of the embryo shown in F.
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pone.0131908.g005: Endogenous gene expression matches the gene-trap GFP expression pattern for agtpbp1 and eps15L1 lines.(A) The GFP pattern in a representative 6 dpf embryo from the agtpbp1fcc301 line. (B-D) WISH of agtpbp1 at 6 dpf. Lateral line cells (*), epiphysis (arrowhead) and nasal pit (arrow) are indicated. (C-D) Magnified views of regions of the embryo shown in B. (E) The GFP pattern in a representative 6 dpf embryo from line eps15L1fcc436. (F-I) WISH of eps15L1 at 6 dpf. Pancreas (under *), kidney (arrowhead) and skin cells (arrows) are indicated. (G-I) Magnified views of regions of the embryo shown in F.

Mentions: Agtpbp1fcc301 gene trap carriers express GFP in the nose, eye, pituary gland, brain, notochord lateral line nerve, lateral line sensory organs and kidney (Fig 5A). WISH analysis of agtpbp1 at 6 dpf shows expression in nose, eye, hypophysis, brain, and lateral line but we could not detect expression in the notochord, lateral line nerve or kidney (Fig 5B–5D). WISH analysis of agtpbp1 at 2 dpf showed expression in the nose, eye, central nervous system and faintly in cells in the caudal hematopoietic region (S8A and S8B Fig), indicating that the GFP pattern is overlapping, although not a perfect reflection of endogenous gene expression. WISH analysis of eps15L1 at 6 dpf showed that this gene is expressed in skin, kidney and pancreas, which overlaps exactly with the GFP expression pattern fcc436 carriers (Fig 5E–5I). Expression of eps15L1 at 2 dpf overlaps the GFP pattern in fcc436 carriers at this age, and was observed in cells in the AGM and CHT regions, although RNA expression was detected throughout the embryo (S8C and S8D Fig). Possible explanations for why the GFP expression does not perfectly reflect endogenous gene expression include that the GFP protein is likely more stable than RNA transcripts, thus the GFP may be retained in cells from expression at earlier developmental stages, or that the level of sensitivity of the UAS-mediated detection system is higher than RNA in situ hybridization. Nonetheless, these data are consistent with agtpbp1 and eps15L1 being the gene trap targets in the fcc301 and fcc436-P1 lines, respectively.


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

Seiler C, Gebhart N, Zhang Y, Shinton SA, Li YS, Ross NL, Liu X, Li Q, Bilbee AN, Varshney GK, LaFave MC, Burgess SM, Balciuniene J, Balciunas D, Hardy RR, Kappes DJ, Wiest DL, Rhodes J - PLoS ONE (2015)

Endogenous gene expression matches the gene-trap GFP expression pattern for agtpbp1 and eps15L1 lines.(A) The GFP pattern in a representative 6 dpf embryo from the agtpbp1fcc301 line. (B-D) WISH of agtpbp1 at 6 dpf. Lateral line cells (*), epiphysis (arrowhead) and nasal pit (arrow) are indicated. (C-D) Magnified views of regions of the embryo shown in B. (E) The GFP pattern in a representative 6 dpf embryo from line eps15L1fcc436. (F-I) WISH of eps15L1 at 6 dpf. Pancreas (under *), kidney (arrowhead) and skin cells (arrows) are indicated. (G-I) Magnified views of regions of the embryo shown in F.
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Related In: Results  -  Collection

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pone.0131908.g005: Endogenous gene expression matches the gene-trap GFP expression pattern for agtpbp1 and eps15L1 lines.(A) The GFP pattern in a representative 6 dpf embryo from the agtpbp1fcc301 line. (B-D) WISH of agtpbp1 at 6 dpf. Lateral line cells (*), epiphysis (arrowhead) and nasal pit (arrow) are indicated. (C-D) Magnified views of regions of the embryo shown in B. (E) The GFP pattern in a representative 6 dpf embryo from line eps15L1fcc436. (F-I) WISH of eps15L1 at 6 dpf. Pancreas (under *), kidney (arrowhead) and skin cells (arrows) are indicated. (G-I) Magnified views of regions of the embryo shown in F.
Mentions: Agtpbp1fcc301 gene trap carriers express GFP in the nose, eye, pituary gland, brain, notochord lateral line nerve, lateral line sensory organs and kidney (Fig 5A). WISH analysis of agtpbp1 at 6 dpf shows expression in nose, eye, hypophysis, brain, and lateral line but we could not detect expression in the notochord, lateral line nerve or kidney (Fig 5B–5D). WISH analysis of agtpbp1 at 2 dpf showed expression in the nose, eye, central nervous system and faintly in cells in the caudal hematopoietic region (S8A and S8B Fig), indicating that the GFP pattern is overlapping, although not a perfect reflection of endogenous gene expression. WISH analysis of eps15L1 at 6 dpf showed that this gene is expressed in skin, kidney and pancreas, which overlaps exactly with the GFP expression pattern fcc436 carriers (Fig 5E–5I). Expression of eps15L1 at 2 dpf overlaps the GFP pattern in fcc436 carriers at this age, and was observed in cells in the AGM and CHT regions, although RNA expression was detected throughout the embryo (S8C and S8D Fig). Possible explanations for why the GFP expression does not perfectly reflect endogenous gene expression include that the GFP protein is likely more stable than RNA transcripts, thus the GFP may be retained in cells from expression at earlier developmental stages, or that the level of sensitivity of the UAS-mediated detection system is higher than RNA in situ hybridization. Nonetheless, these data are consistent with agtpbp1 and eps15L1 being the gene trap targets in the fcc301 and fcc436-P1 lines, respectively.

Bottom Line: An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults.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.

View Article: PubMed Central - PubMed

Affiliation: Blood Cell Development and Function Program, Fox Chase Cancer Center, Temple University Health System, 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.

No MeSH data available.


Related in: MedlinePlus