Figure 4.
Defective thymus migration of hematopoietic progenitors in ccr9a mutant embryos. A, WISH of ccr9a in the CHT of ikzf1Δ4 + 3/Δ4 + 3 mutant embryos and their siblings. B and C, ChIP-qPCR analysis (C) (mean ± S.D.; ccr9a-1F/1R, IgG: 1.00 ± 0.03, anti-HA: 4.73 ± 0.51; ccr9a-2F/2R, IgG: 1.00 ± 0.64, anti-HA: 15.13 ± 3.84; ccr9a-CF/CR, IgG: 1.00 ± 0.33, anti-HA: 1.14 ± 0.35) of the binding sites (green) in the ccr9a promoter for Ikzf1 (B). CF, control forward primers; CR, control reverse primers. D, luciferase assay showing the activities of ccr9a promoter (with the binding site (BS) and mutant binding site (MBS)) when regulated by the Ikzf1 protein (full-length CDS) (mean ± S.D.; ccr9a promoter: 1.00 ± 0.06, n = 3; ccr9a promoter + ikzf1 CDS: 3.07 ± 0.29, n = 3; ccr9a promoter (MBS1) + ikzf1 CDS: 2.17 ± 0.04, n = 3; ccr9a promoter (MBS2) + ikzf1 CDS: 2.51 ± 0.34, n = 3; ccr9a promoter (MBS1+MBS2) + ikzf1 CDS: 1.79 ± 0.04, n = 3). EV, pCS2 empty vector. E and F, WISH of tcrb and rag1 (E), and cmyb (F) in ccr9aΔ10/Δ10 mutant larvae and their siblings. G, fluorescence image of thymi of ccr9aΔ10/Δ10 mutant embryos and their siblings. H, calculation of spherical coro1a-Kaede+ cells entering the thymus per hour (from 60–72 hpf; mean ± S.D.; Sib: 0.63 ± 0.15, n = 6; ccr9aΔ10/Δ10: 0.09 ± 0.06, n = 5). See also Movies S3 and S4. I, fluorescence images indicating labeled coro1a-Kaede+ cells in thymi of ccr9aΔ10/Δ10 mutant embryos and their siblings. J, calculation results of I (mean ± S.D.; Sib: 11.27 ± 5.06, n = 26; ccr9aΔ10/Δ10: 3.56 ± 2.04, n = 18). The blue arrowheads indicate the WISH signals (A and F). The red (E and F) and white (G and I) circles represent the thymi. The values in the bottom right corner in A, E, F, and I indicate counts with a typical appearance as presented (first number) in the total number of examined samples (last number). OV, otic vesicle. Scale bars = 50 μm. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.