Expression of flk1 in clo¹⁷² and clo^s5 mutants.

(A–I) Whole-mount in situ hybridization of flk1 expression at 19.5 hpf in sibling (A), clo¹⁷² mutant (B) and clo^s5 mutant (C) embryos, 1 dpf stage in sibling (D), clo¹⁷² mutant (E) and clo^s5 mutant (F) embryos, and 2 dpf stage in sibling (G), clo¹⁷² mutant (H) and clo^s5 mutant (I) embryos. Embryos are shown with anterior to the left and dorsal up. Inserts are high magnification (20×) of the corresponding boxed regions. DA: dorsa aorta; PCV: posterior cardinal vein; DLAV: dorsal longitudinal anastomotic vessels.

Dynamic βe1 expression during primitive hematopoiesis in clo¹⁷² and clo^s5 mutant embryos.

(A–I) Whole-mount in situ hybridization of βe1 expression in sibling embryos at 14 hpf (A), 1 dpf (D) and 36 hpf (G), and clo¹⁷² mutant embryos at 14 hpf (B), 1 dpf (E) and 36 hpf (H), and clo^s5 mutant embryos at 14 hpf (C), 1 dpf (F) and 36 hpf (I). Embryos are shown with anterior to the left and dorsal up. PLM: posterior lateral mesoderm; ALM: anterior lateral mesoderm; ICM: intermediate cell mass; VDA: ventral wall of dorsa aorta.

Primitive Myelopoiesis in clo¹⁷² and clo^s5 mutant embryos.

(A–I) Myeloid lineage marker l-plastin expression at 19.5 hpf(A: the left arrow show anterior cephalic mesoderm, B–C), 1 dpf stage (D: the left arrow show anterior cephalic mesoderm, E–F), and 36 hpf stage (G: the left arrow show anterior cephalic mesoderm H–I) in sibling, clo¹⁷² and clo^s5 mutant embryos. Embryos are shown with anterior to the left and dorsal up. Inserts are high magnification (20×) of the corresponding boxed region (the right arrow show tail region).

Definitive hematopoiesis in clo¹⁷² and clo^s5 mutant embryos.

(A–C) stem cell marker c-myb expression at 36 hpf and 3 dpf stage in wild type (A: arrow show VDA region; D: arrow show PBI region), clo¹⁷² (B, E) and clo^s5 mutant (C, F) embryos.(G–I). WISH of βe1 expression at 4 dpf stage in sibling (G: arrow show PBI region), clo¹⁷² (H) and clo mutant (I) embryos. (J–L) WISH to detect l-plastin expression at 4 dpf stage in sibling (J: arrow show PBI region), clo¹⁷² (K) and clo^s5 mutant (L) embryos. (M–O)WISH of T lymophcyte marker rag1 expression at 4 dpf stage in sibling (M: arrow show thymus), clo¹⁷² (N) and clo^s5 mutant (O) embryos. Embryos are shown with anterior to the left and dorsal up. Inserts are high magnification (20×) of the corresponding boxed regions. VDA: ventral wall of dorsa aorta; PBI: posterior blood island.

Analysis of the origin of definitive myeloid cells in clo¹⁷² mutant.

(A, B) lyc expression pattern at 3 dpf runx1/clo¹⁷² double mutant (A). WISH of lyc expression in siblings (A–i), clo¹⁷² mutant (A–ii), clo¹⁷² (-/-) runx1 (+/-) ^w84x (A-iii), double homozygous mutant (A–iv). Histogram of lyc⁺ cells number (means) in siblings(B, purple column), clo¹⁷² mutant (B, green column), clo¹⁷² (-/-) runx1 (+/-)^w84x (B, pink column) and double homozygous mutant (B, blue column). There are significant differences between double mutant and other groups (*p<0.05), but no difference between clo¹⁷² mutant and clo¹⁷² (-/-) runx1 (+/-) ^w84x group (p>0.05). The corresponding case numbers were shown by n in column.

Dynamic morphological changes of clo¹⁷² and clo^s5 mutant. (A–F) Lateral view of morphological changes 30 hpf stage of sibling (A), clo¹⁷² (B), clo^s5 mutant (C) and 2 dpf stage of sibling (D), clo¹⁷² (E: arrow show edema heart and red blood cell in VDA region), clo^s5 mutant (F: arrow show edema heart).

Expression of lyc during primitive hematopoiesis in clo¹⁷² and clo^s5 mutant embryos. (A–F) Whole-mount in situ hybridization of lyc expression at 19.5 hpf (A: arrow show anterior cephalic mesoderm, B–C), 1 dpf stage (D: the left arrow show anterior cephalic mesoderm, E–F) in sibling, clo¹⁷² mutant and clo^s5 mutant embryos. Embryos are shown with anterior to the left and dorsal up. Inserts are high magnification (20×) of the corresponding boxed regions.

Definitive myelopoiesis and rag1 expression pattern in clo¹⁷² and clo^s5 mutant embryos. l-plastin and lyc expression at 3 dpf in sibling(A, D), clo¹⁷² (B, E), clo^s5 mutant (C, F). rag1 expression at 5 dpf in sibling(G), clo¹⁷² (H), clo^s5 mutant (I). Inserts are high magnification (20×) of the corresponding boxed regions (the right arrow show tail region).