The mutant^cas008 line showed defective definitive hematopoiesis. WISH analysis of myb(A,A′), ae1-globin(B,B′), lyz(C,C′), mpx(D,D′), and rag1(E,E′) expression in sibling (A–E) and mutant^cas008(A′–E′) embryos at 5 dpf. The penetrance of the indicated phenotype is shown in the bottom right of each panel. Black arrows indicate the position of CHT, while red arrows and circles show the position of thymus and kidney, respectively.

The mutation was mapped to a point mutation in gene gemin5. (A) The alignment of WT (underlined) and NC_007132.7: g.36404684 C > T (gemin5⁰⁰⁸) sequences are listed. The mutation by ENU is indicated in red (a C to T nonsense mutation causes an immediate stop codon in gemin5 in mutant^cas008). (B) According to the stop codon in the genome, SMART software was used to predict the structure of Gemin5^WT and XP_001339880.4:p.Gln303_His1440del (Gemin5⁰⁰⁸) presumed protein. The molecular sizes of the presumed protein are indicated on the right. (C) WISH analysis with myb probe in the CHT region (at 6 dpf) of sibling and gemin5⁰⁰⁸ with transient transgenesis of vector (only plasmid backbone) or ubi: gemin5 (#1, The construction of the plasmid is shown below).

Decreased HSPCs proliferation in mutant gemin5^cas008. (A,B) Representative confocal images of double staining of pH3 protein (green) and myb RNA (red) in the CHT at 4 dpf (A) and 5 dpf (B). Arrows point at proliferative HSPCs. Scale bars, 50 μm. (C,D) Percentage of proliferative HSPCs (pH3+myb+)/ total HSPCs (myb+) in sibling and gemin5^cas008 embryos at 4 dpf (C) and 5 dpf (D). (C)t = 4.202, df = 12, and P = 0.0012. (D)t = 10.96, df = 10, and P < 0.0001.