Fig 2

Fig 2

(A) Expression of HSPC markers runx1 and cmyb in control and rab5c morphants examined by WISH. The red arrowheads denote HSPCs. Scale bar, 100 μm. (B) Quantification of WISH results. Error bars, mean ± SD, ***P < 0.001. (C) Expression of differentiation markers: erythroid marker gata1, neutrophil marker lyz, myeloid marker pu.1, and lymphoid marker rag1 in the control and rab5c morphants at 4 dpf examined by WISH. Scale bar, 100 μm. (D) Quantification of panel C. Error bars, mean ± SD, ***P < 0.001. (E) Statistical analysis of the WISH. Error bars, mean ± SD, ***P < 0.001. (F) Confocal imaging shows the kdrl⁺cmyb⁺ definitive hematopoietic precursors in the VDA region of control and rab5c morphants at 36 hpf. White arrowheads denote precursors. Scale bar, 100 μm. (G) Quantification of kdrl⁺cmyb⁺ precursors. Error bars, mean ± SD, ***P < 0.001. (H) Confocal imaging shows that there are less CD41⁺ HSPCs (green) in the CHT (box area) of rab5c morphants compared with control at 2 dpf. Scale bar, 100 μm. (I) Quantification of CD41⁺ HSPCs. Error bars, mean ± SD, ***P < 0.001. (J) Expression of HE marker gfi1a in control and rab5c morphants examined by WISH. Scale bar, 100 μm. (K) Quantification of the gfi1a positive cells. Error bars, mean ± SD, ***P < 0.001. (L) Generation of rab5c frameshift mutant using the CRISPR/Cas9 technique. Location and sequence of the target site are exhibited. rab5c wild type and mutant sequences are listed below. (M) Expression of runx1 in rab5c wild type and mutant at 36 hpf examined by WISH. Scale bar, 100 μm. (N) Quantification of the runx1 positive cells. Error bars, mean ± SD, ***P < 0.001. The P values in this figure were calculated by Student t test. The underlying data in this figure can be found in S1 Data. CHT, caudal hematopoietic tissue; dpf, days post fertilization; DA, dorsal aorta; HE, hemogenic endothelium; HSPC, hematopoietic stem and progenitor cell; hpf, hours post fertilization; PAM, protospacer adjacent motif; VDA, ventral wall of the dorsal aorta; WISH, whole-mount in situ hybridization .