Reciprocal crossovers and gene conversion.

<p>(A) An RCO is depicted between chromatids of two homologous chromosomes. One segregation pattern results in daughter cells that have become homozygous for the sequence distal to the crossover site. (B–D) A close-up view of the region outlined by the dotted box, showing different gene conversion tract configurations detectable using markers <i>a</i> through <i>d</i>. (B) No conversion tract, either because there was no gene conversion or the tract was too small to be detected with the markers available. All markers are still present in a 2∶2 ratio. (C) A typical gene conversion event produces a tract that alters some of the markers (<i>b</i> and <i>c</i>) to a 3∶1 ratio. Note that conversion tracts can only be detected if both reciprocal products (i.e., both daughter cells) are recovered and analyzed, as done by Lee et al. (D) Lee et al. observed some tracts that were wholly or partially 4∶0. In the example shown here, marker <i>b</i> has segregated 4∶0, but marker <i>c</i> has segregated 3∶1; this is therefore a 4∶0/3∶1 hybrid gene conversion tract.</p

Decreased expression of lymphoid markers in gene-trap mutant embryos.

<p>(A-F) Whole mount RNA in situ hybridization (WISH) of <i>lck</i> in 5 dpf larvae from the lines <i>fcc143</i> (143) (A-B), <i>agtpbp1</i>^{<i>fcc301</i>} (301) (C-D) and <i>eps15L1</i>^{<i>fcc436-P1</i>} (436) (E-F). Representative embryos displaying no GFP (A, E) or normal expression of <i>lck</i> (C) are compared to siblings with strong (str) GFP (B, F) or low levels of <i>lck</i> expression (D). In line <i>agtpbp1</i>^{<i>fcc301</i>}, the GFP expression levels varied, making it difficult to distinguish heterozygous and homozygous carriers. The number of embryos that showed the representative phenotype is indicated in panels C and D.</p

Patterns of GFP expression in <i>GBT-B4</i> gene trap lines that include hematopoietic tissues.

<p>Lateral views of embryos at 48 hpf or 6 dpf are shown. The <i>Tg(GBT-B4)fcc</i> line number is indicated to the left of the panels. The embryo age is indicated. CHT = caudal hematopoietic tissue; AGM = aorta-gonad-mesonephros. Note that the lines are grouped by hematopoietic expression patterns, but the embryos can express GFP in additional tissues.</p

Summary of lines with embryonic marking of hematopoietic tissue and identification of the disrupted genes.

<p>*<i>GBT-B4</i> = <i>Gt(LOXP-GAL4-VP16-FRT</i>, <i>syUAS</i>:<i>EGFP-FRT-LOXP)</i></p><p>Summary of lines with embryonic marking of hematopoietic tissue and identification of the disrupted genes.</p

The <i>GBT-B4</i> genetic screen approach and results.

<p>(A) Design features of the <i>GBT-B4</i> gene trap and the nucleotide sequence of the 14XUAS used in the parental <i>GBT-B1</i> and the 4.5xUAS syUAS engineered of <i>GBT-B4</i>. The consensus Gal4 binding site based on the 14XUAS is in red bold letters, sequences based on the Saccharomyces UAS are in bold green. The core Gal4 binding site CGGN₁₁CCG is highlighted in yellow. The non-consensus nucleotide in the 4^th binding site of the syUAS is in lower case (t instead of G). (B) Diagram of the genetic screen; arrows indicate the workflow. (C) Summary of the screen results.</p

Morpholino knockdown of <i>agtpbp1</i> and <i>eps15L1</i> results in decreased lymphoid <i>rag1</i> expression.

<p>(A-B) WISH of <i>rag1</i> in 4 dpf control (A) and <i>agtpbp1</i> (B) morpholino-injected embryos. (C) RT-PCR of <i>agtpbp1</i> and <i>ß-actin</i> in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to <i>ß-actin</i> is indicated. (D-E) WISH of <i>rag1</i> in 4 dpf control (A) and <i>eps15L1</i> (B) morpholino-injected embryos. (C) RT-PCR of <i>eps15L1</i> and <i>ß-actin</i> in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to <i>ß-actin</i> is indicated. Quantitation is in arbitrary units (A.U.), and relative to wild-type level which is set at 1. Head region of the embryos is shown in lateral views, anterior to the left. P values were determined using Fisher’s exact test.</p

Identification of GFP populations that express T and B cell markers in young adult fish.

<p>(A) Examples of scatter plots from fluorescence-activated cell sorting (FACS) of GFP+ cells from the indicated <i>Tg(GBT-B4)fcc</i> lines. Plots show GFP versus forward scatter (FSC, indicates cell size). The AB wildtype is a negative control. The line name and percent positive GFP cells out of the total events is indicated for each plot. (B) RT-PCR analysis of <i>igH-</i>μ, <i>lck</i> and <i>ß-actin</i> in GFP⁺ cells from the indicated Tg(GBT-B4)fcc lines. Lanes lacking an <i>actin</i> signal are not shown; vertical separation indicates independent experiments. Duplicates of fcc301 are shown (B-C). (C) Quantification of <i>igH-</i>μ and <i>lck</i> expression in GFP⁺ cells from the indicated lines. A.U. = arbitrary units. The scoring scale is listed below the results.</p

Mutagenesis Screen Identifies <i>agtpbp1</i> and <i>eps15L1</i> as Essential for T lymphocyte Development in Zebrafish

<div><p>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 (<i>Danio rerio</i>, 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, <i>agtpbp1</i> and <i>eps15L1</i>. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of <i>agtpbp1</i> and <i>eps15L1 </i>in T cell development.</p></div

Endogenous gene expression matches the gene-trap GFP expression pattern for <i>agtpbp1</i> and <i>eps15L1</i> lines.

<p>(A) The GFP pattern in a representative 6 dpf embryo from the <i>agtpbp1</i>^{<i>fcc301</i>} line. (B-D) WISH of <i>agtpbp1</i> 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 <i>eps15L1</i>^{<i>fcc436</i>}. (F-I) WISH of <i>eps15L1</i> 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.</p