Foxp3 phylogeny and identification of foxp3a:EGFP-positive lymphocytes. (A) Phylogenetic relationship of zebrafish Foxp3 proteins to Foxp3 proteins from other species (human, Homo sapiens, H.s.; mouse, Mus musculus, M.m.; frog, Xenopus laevis, X.l.; pufferfish, Tetraodon nigroviridis, T.n.) and other human Foxp family proteins. Unrooted neighbor-joining best trees were produced using MacVector v12.5.1. Sequences were aligned by ClustalW. Distance was absolute, and gaps were distributed proportionally. GenBank accession nos. for sequences used were D.r. Foxp3a (NP_001316496), D.r. Foxp3b (XM_021478427), H.s. Foxp1 (NP_001231745), H.s. Foxp2 (NP_055306), H.s. Foxp3 (ABQ15210), H.s. Foxp4 (NP_001012426), M.m. Foxp3 (NP_001186277), X.l. Foxp3 (NP_001121199), and T.n. Foxp3 (ADD91631). (B) qRT-PCR of foxp3 paralogs in wild-type AB WKM relative to nonhematopoietic fin tissue. Error bars indicate SEM; n = 3. (C) Flow cytometry analysis of WKM from representative AB and Tg(foxp3a:EGFP) animals. Gates of major hematopoietic lineages are indicated at right. (D) Percentages of EGFP-positive cells in each gate are shown, and data were used to calculate fold differences of EGFP-positive cells in Tg(foxp3a:EGFP; n = 5) and Tg(foxp3a:EGFP); rag1(lf) (n = 5) versus intrinsically fluorescent cells in wild-type AB animals (n = 5). Two-tailed Student’s t test, Tg(foxp3a:EGFP) versus Tg(foxp3a:EGFP); rag1(lf), **, P < 0.001. (E) Cytological stains of sorted foxp3a:EGFP-positive cells compared with sorted lck:EGFP-positive lymphocytes. Individual cells were extracted and aligned for comparative purposes. (F) Flow cytometry of WKM from a representative Tg(foxp3a:EGFP); rag1(lf) animal showing absence of the EGFP-positive population. (G) qRT-PCR of foxp3 paralogs in foxp3a:EGFP-positive cells from WKM compared with bulk WKM lymphocytes. †, Expression of foxp3b was below the limit of detection in foxp3a:EGFP-positive and bulk lymphocytes. Error bar indicates SEM; n = 3.