Development of the leukocyte lineages in ptpn6 mutants. (A-H) A panel of in situ hybridization markers for leukocyte lineages was used on 5 dpf WT and ptpn6−/− embryos. The CHT is indicated by brackets and the thymus by white arrows. The number of embryos showing the depicted pattern/total number of embryos is shown in the bottom right corner. The localization of L-plastin (lcp1), pan-leukocyte marker (A,B); ikaros, lymphoid progenitor marker (C-F); and rag1, lymphocyte marker (G,H) was analyzed. Representative lateral views of the thymus are shown, with close ups of the thymus in the inset (G,H). (I) Quantification of rag1-positive area. One-way ANOVA no post hoc test because no significance was detected. A.U., arbitrary units. (J,K) Localization of csf1r, a macrophage marker. J′,K′ show close ups of the CHT. (L-O) Sudan Black (SB) staining of neutrophils in two parts of the embryo at 5 dpf. L′,N′ show magnifications of the boxed areas in L,N. (P,Q) SB-positive cells were counted in the head (mainly primitive wave) and CHT (mainly definitive wave) at 3 and 5 dpf. All embryos were genotyped after in situ hybridization or SB staining and imaging by PCR and sequencing. Statistical comparisons were performed by one-way ANOVA followed by Tukey's multiple comparisons for both areas per time point separately. ns, not significant; **P<0.01; ****P<0.0001. Error bars show the s.e.m. Scale bars: 0.2 mm (A-H); 0.5 mm (J,K); 0.2 mm (L-O).
