Fig 1.
The GBT-B4 genetic screen approach and results.
(A) Design features of the GBT-B4 gene trap and the nucleotide sequence of the 14XUAS used in the parental GBT-B1 and the 4.5xUAS syUAS engineered of GBT-B4. 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 4th 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.
Fig 2.
Patterns of GFP expression in GBT-B4 gene trap lines that include hematopoietic tissues.
Lateral views of embryos at 48 hpf or 6 dpf are shown. The Tg(GBT-B4)fcc 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.
Table 1.
Summary of lines with embryonic marking of hematopoietic tissue and identification of the disrupted genes.
Fig 3.
Identification of GFP populations that express T and B cell markers in young adult fish.
(A) Examples of scatter plots from fluorescence-activated cell sorting (FACS) of GFP+ cells from the indicated Tg(GBT-B4)fcc 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 igH-μ, lck and ß-actin in GFP+ cells from the indicated Tg(GBT-B4)fcc lines. Lanes lacking an actin signal are not shown; vertical separation indicates independent experiments. Duplicates of fcc301 are shown (B-C). (C) Quantification of igH-μ and lck expression in GFP+ cells from the indicated lines. A.U. = arbitrary units. The scoring scale is listed below the results.
Fig 4.
Decreased expression of lymphoid markers in gene-trap mutant embryos.
(A-F) Whole mount RNA in situ hybridization (WISH) of lck in 5 dpf larvae from the lines fcc143 (143) (A-B), agtpbp1fcc301 (301) (C-D) and eps15L1fcc436-P1 (436) (E-F). Representative embryos displaying no GFP (A, E) or normal expression of lck (C) are compared to siblings with strong (str) GFP (B, F) or low levels of lck expression (D). In line agtpbp1fcc301, 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.
Fig 5.
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.
Fig 6.
Morpholino knockdown of agtpbp1 and eps15L1 results in decreased lymphoid rag1 expression.
(A-B) WISH of rag1 in 4 dpf control (A) and agtpbp1 (B) morpholino-injected embryos. (C) RT-PCR of agtpbp1 and ß-actin in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to ß-actin is indicated. (D-E) WISH of rag1 in 4 dpf control (A) and eps15L1 (B) morpholino-injected embryos. (C) RT-PCR of eps15L1 and ß-actin in pooled control or morphant embryo samples. Quantitation of the normal transcript band normalized to ß-actin 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.