T-cell development is impaired in zebrafish foxn1 morphants. (A and B) The endogenous expression level of foxn1 transcript and the encoded protein in zebrafish foxn1 morphants at 4 dpf detected by WISH (A) and Western blot (B). Ba, Western blot; Bb, Western blot results were quantified by using Quantity 1 software. (C) The rag2:dsRed expression was abolished in foxn1 morphants at 5 dpf. (D) The expression of lymphocyte markers, including rag1, il7r, and ikaros, was down-regulated in the thymus in zebrafish foxn1 morphants. Anterior to the left and dorsal to the top; circles mark the thymus. (E) qRT-PCR results showing that the expression of rag1, il7r, and ikaros is down-regulated in zebrafish foxn1 morphants (mean ± SD, t test, **P < 0.01, n = 3).

Four genes are specifically decreased in the thymus in zebrafish foxn1 morphants. (A–C) The expression level of cbfβ, cdca7, mcm2, and runx3 in the thymus in zebrafish foxn1 morphants using qRT-PCR (A; mean ± SD, Student′s t test, *P < 0.05, **P < 0.01, n = 3), WISH (B), and Western blot (C), respectively. Ca, Western blot; Cb, Western blot results were quantified by using Quantity one software. Anterior to the left and dorsal to the top; circles mark the thymus.

T-cell development is impaired in zebrafish cdca7 and mcm2 morphants. (A) The protein expression of Cdca7 and Mcm2 in the zebrafish cdca7 and mcm2 morphants detected by using Western blot. (B) The expression of T-cell markers (rag1, il7r, and ikaros) in zebrafish cdca7 and mcm2 morphants by qRT-PCR (mean ± SD, Student’s t test, **P < 0.01, n = 3). (C) The expression of rag1, il7r, ikaros, and foxn1 in zebrafish cdca7 and mcm2 morphants by WISH. Anterior to the left and dorsal to the up; circles mark the thymus.

The expression of rag1 and ikaros can be rescued in foxn1 morphants coinjected with mRNAs. (A) The expression of rag1 and ikaros in foxn1 morphants coinjected with mRNAs using WISH. (B) The expression of ikaros in foxn1 morphants coinjected with mRNAs using qRT-PCR (mean ± SD, n = 3). (C) The protein level of Foxn1 in foxn1 morphants coinjected with mRNAs.

mcm2 is a direct target of Foxn1. (A) Predicted Foxn1 binding site and ChIP-PCR analysis of the Foxn1 binding to the promoter region of mcm2. The consensus site was marked by colored letters. (B) qRT-PCR analysis of Foxn1 binding to the promoter region of mcm2 in control and foxn1 morphants (mean ± SD, n = 3). (C) The mcm2 promoter and the mcm2p truncated constructs, and the luciferase reporter assay (mean± SD, n = 3). HEK293 cells were cotransfected by the Renilla reporter plasmid and the mcm2 promoter construct together with pCDNA3.3(+)-foxn1. Luciferase assays were determined by using the Dual-Luciferase Reporter Assay System (Promega). 2F/2R stand for a pair of gene specific primers spanning the Foxn1 binding site; CF/CR stand for control primers. The results indicate that the functional consensus Fox binding sites of the mcm2 promoter are positively regulated by Foxn1 in a dose-dependent manner.

TEC proliferation defects in foxn1 and mcm2 morphants by BrdU labeling. (A) Reduced anti–BrdU-positive cells in foxn1- and mcm2 morphants by BrdU labeling on whole mount. Anti-BrdU positive cells were quantified in the thymus and the head region without the thymus of controls (n = 15), foxn1 morphants (n = 23), and mcm2 morphants (n = 19). Green, anti–BrdU-positive cells; blue, DAPI staining. Circles indicate thymus. (B) Reduced anti–BrdU-positive cells in control, foxn1 morphants, and mcm2 morphants by BrdU labeling on thymus sections. Dotted lines indicate the thymus area. Section thickness, 10 µm. The average number of anti–BrdU-positive cells per section was quantified. (C) Immunofluorescence on thymus sections with anti–Pan-CK staining, which clearly showed reduced number of TECs and smaller size of thymus in foxn1 and mcm2 morphants, compared with controls. Green, Pan-CK staining; blue, DAPI staining. (D) A proposed model of foxn1 functions in thymus development. Dashed square, published data; red arrows, this work.

Hematopoietic stem cells, T cells, and parathyroid development in foxn1 morphants. (A) Population of cmyb:GFP positive T cells in the thymus was much reduced, whereas numbers of cmyb:GFP positive hematopoietic progenitors in the pronephros and caudal hematopoietic tissue at 4 dpf were not much altered in foxn1 morphants. The thymus and pronephros in Upper were marked with circles in yellow and red, respectively. Arrows in Lower mark hematopoietic progenitors. (B) Expression of a parathyroid marker, gcm2, was unchanged in foxn1 morphants.

The expression of epithelial markers and chemokine ligand/receptor in zebrafish foxn1 morphants at 4 dpf. (A) The expression of epcam and hoxa3a, in zebrafish foxn1 morphants, were examined on transverse sections after WISH. Note that expression of these genes in the thymus area was relatively normal in foxn1 morphants. The thymus was marked by using dashed lines. (B) The expression of chemokine ccl25a and receptor ccr9b in zebrafish foxn1 morphants. Note that expression of ccl25a and ccr9b was severely reduced in foxn1 morphants. Anterior to the left and dorsal to the up; the thymus was marked with circles.

Expression profiling and verification of microarray results using semiquantitative RT-PCR and WISH. (A) Heatmap analysis for microarray data at 2 dpf (a) and 4 dpf (b). (B) Overview of the microarray data. (C) Verification of microarray results and a list of down-regulated genes from microarray data were chosen for further validation by semiquantitative RT-PCR (C) and transverse sectioning after WISH (D). Dashed lines mark the thymus region.

Genes expression in zebrafish cloche mutant, foxn1 morphants, mouse thymus, and TEC cell lines. (A) The expression of six genes (foxn1, rag1, cbf&betal, cdca7, mcm2, and runx3) in zebrafish cloche mutants by WISH. Anterior to the left and dorsal to the up; circles mark the thymus. (B) Transverse sectioning after WISH showed that the expression of cbfβ, cdca7, mcm2, and runx3 in the thymus was reduced in foxn1 morphants compared with controls. Dashed lines mark the thymus. (C) The expression of mcm2, cdca7, runx3, cbfβ, and actin in mouse thymus and TEC cell lines (m4c18 and m1c6) using semiquantitative RT-PCR.

The expression of T-cell markers and TEC markers in mcm2 and cdca7 morphants. (A) The expression of T-cell markers, including cbfb, and runx3, in mcm2 and cdca7 morphants. (B) The expression of foxn1 was reduced in mcm2 and cdca7 morphants at 5 dpf. (C) Verification of mcm2- and cdca7 splice MOs using RT-PCR. (D) The expression of rag1 and ikaros in mcm2- and cdca7 morphants injected with splice MOs. Anterior to the left and dorsal to the up; circles mark the thymus.

The impaired T-cell development in mcm2 morphants was not due to altered apoptosis. (A) TUNEL assay in control, foxn1 morphants, mcm2 morphants, and p53+mcm2 morphants. (B) The expression of rag1 and ikaros in mcm2 morphants and p53+mcm2 double morphants using WISH. (C) The expression of ikaros in mcm2 morphants and p53+mcm2 double morphants using qRT-PCR (mean ± SD, Student’s t test, **P < 0.01, n = 3).

The structure of the thymus in control, foxn1 morphants, and mcm2 morphants. (A) The histology of the thymus in control, foxn1 morphants, and mcm2 morphants at 4 dpf using toluidine blue staining on semithin sections. The yellow dotted circles mark the thymus. (B) The ultrastructure of the thymus in control, foxn1 morphants, and mcm2 morphants at 4 dpf using transmission electron microscopy. T lymphocytes were round-shape and the nucleus staining was dense, whereas TECs were less stained and were reticular-shape in controls but irregular in morphants. L stands for the lymphocytes and E stands for the TEC. (Scale bars: Upper, 2 μm; Lower, 1 μm.) (C) The number of TEC and lymphocytes in control, foxn1 morphants and mcm2 morphants at 4 dpf on semithin section of the thymus. The ratio of numbers of TECs or T cells in individual morphant versus control thymus sections are shown as a percentage.