Fig. 2

T-cell maturation is impaired in rb1-deficient embryos. (A) Expression of ikaros, lck and rag1 in the thymus (broken line) of siblings and rb1 mutants at 5?dpf (upper panels). Embryos for whole-mount in situ hybridization were obtained from an incross of genotyped heterozygous rb1 mutants. Scale bars: 100??m. The positive signal areas were analysed using Image-Pro Plus (lower panel) (***P<0.001; mean±s.e.m.; n=16). (B) Confocal images of rag2:DsRed cells in the thymus of the siblings and rb1^smu8/smu8 mutants from 4?dpf to 8?dpf (upper panels). Scale bars: 50??m. Quantification of T-cell number in sibling embryos and rb1^smu8/smu8 mutants (lower panel) (***P<0.001; mean±s.e.m.; n=10). (C) Schematic of the lateral view of a zebrafish indicating the region of the thymus excised for RNA extraction (red dot). qPCR analysis of rag2, cd4, foxn1 and myb in the thymus of siblings and rb1-deficient embryos at 5?dpf (mean±s.e.m.; **P<0.01, *P<0.05, ns, not significant; n=30). (D) Whole-mount in situ hybridization of foxn1 in the thymus (broken line) of siblings and rb1^smu8/smu8 mutants at 5?dpf. Scale bars: 100??m. (E) Whole-mount in situ hybridization of cmyb in the thymus of the siblings and rb1^smu8/smu8 mutants at 3?dpf. Scale bars: 20??m. (F) Whole-mount in situ hybridization of rag1 in the thymus (broken line) at 5?dpf after injecting with control and pTol-lck:rb1 plasmid. Scale bars: 100??m.