Wnt And Notch Signal Pathway Regulating Hematopoiesis During Hemotapoietic Development

【Objectives】To study the mechanism of wnt and notch signal pathway regulating hematopoiesis during hematopoietic development.【Methods】we separated murine c-kit~+ hematopoietic progenitor cells (HPCs) from different sources which stand for different stages and different times of hematopoiesis by MACS system. The sources include aorta-gonad-mesonephros (AGM) region at E10.5 and E11.5, fetal liver at E12.5, E14.5d, E16.5d, E18d, and bone marrow of adult murine. The purity of the c-kit~+ cells was detected by flow cytomrtry. The gene expressions of Wnt and Notch pathway members were detected by quantitative PCR. The expression of Wnt3a and Notch1 proteins were detected by immunostaining. E10.5 AGM-, E14.5 FL- and adult BM-derived c-kit~+ cells were cultured in vitro. After seven days, the total number of cells were counted, the expression of c-kit phenotype was detected by flow cytometry and the progenitor function were assessed by CFU assays.【Results】Using the MACS system, the purity of separated c-kit~+ cells can reach at more than 90%. Wnt and Notch signal pathway showed highly variable expression patterns at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived c-kit~+ cells, comparatively, Notch was highly expressed in AGM and BM derived c-kit~+ cells. Immunostaining analysis of E10.5 AGM derived c-kit~+ cells showed very strong staining for Wnt3a and Notch1. In FL-derived c-kit~+ cells, the expression of Notch1 was much lower compared with AGM. In BM derived c-kit~+ cells, the expression of Wnt3a was much lower and Notch1 was much higher compared with c-kit~+ cells derived from FL. After 7days of culture, AGM-derived c-kit~+ cells produced the highest number of hematopoietic cells while BM-derived c-kit~+ cells produced the lowest number of hematopoietic cells(p<0.05). And a higher percentage level of c-kit~+ cells was detected in AGM- and BM-derived c-kit~+ cells culture system compared to FL (p<0.05).【Conclusion】Genes related to the Wnt signaling pathway were up-regulated in E10.5 AGM and E14.5 FL corresponding to the inherent proliferation potential of hematopoietic progenitors whereas genes related to the Notch signaling pathway were identified as up-regulated in E10.5 AGM and bone marrow (BM) coincides with the maintenance of undifferentiation state of hematopoietic progenitors. Our findings suggest that Wnt and Notch signalings are integrated and selectively regulating hematopoiesis. The spatial and temporal balance between Wnt and Notch signaling orchestrates the precise progression of hematopoietic progenitors.