Exploration Of Hematopoietic Amplification Region Of Mouse Embryo And Molecule Mechanism In HSCs' Expansion During E10～E12

PartⅠ[Objective] To investigate the anatomic structure of mouse hematopoietic region during AGM period of E10-E12.[Methods] Matings for embryo generation were timed and the day of vaginal plug detection was designated as embryonic day 0 (E0). The mouse embryo of E10, E10.5, E11, E11.5 and E12 were collected. The embryoemma and the placenta were removed. After fixed in formalin for 3 hours, dehydrated by gradient alcohol, embedded in paraffin according to standard protocols, sliced through sagittal plane and dyed by HE, the histological anatomy of the mouse hematopoietic region was observed under a microscope.[Results] The typical tubiform structure constituted by the aorta, gonad and mesonephron region was not observed in the dorso- abdominalis through the sagittal plane from E10 to E12. The embryo heart had developed, presenting the globular appearance with cancellous spatial structure consisted of cadiocyte and superficial slender endotheliocyte linked with each other at E10. The circulation had established at E10 from the slice of the mouse embyo. It is evident that a few cyloid mononuclear HSCs with the affluent kytoplasm had emerged in the heart and the blood vessel. There was no significance difference in the histological section of embryo heart between E10.5 and E11. But the quantity of hematopoietic cells was obviously increased at El 1, and they were easily seen to touch with the endothelitcytes of blood vessel and the heart. As the embryo developed, the shape of the heart changed quickly, with network structure diminished gradually. And at E12 the heart had becomed to the hollow organ, owning the characteristic of adult heart. The fetal liver had developed at E10, which was the substantial structure and composed with fetal liver cells arranged tightly. The blood vesse of fetal liver interior increased gradually, as the fetal liver developed. [Conclusion] The typical AGM region was not discovered through serial sections of mouse embryo's median sagittal plane and HE staining. The embryo heart emerged as complicate network structure at E10, with the endotheliocytes covering the cadiocytes, which may provide the favorable miroenvironment for the development and proliferation of HSCs through the gigantic surface area.PartⅡ[Objective] To research the characteristics of endotheliocyte in the mouse embryo heart during E10～E12 and molecule mechanism related to the hematopoietic development. [Methods] The histological sections of E10 to E12 were immunostained with anti-mouse CD34, Notch1 and Jagged1, and examined under a microscope. The c-kit+ cells in the heart and fetal liver were sorted through immunomagnetic beads and the expression of Notch1 protein was detected by immunofluorescence technique. The mononuclear cells (MNC) of the heart, fetal liver and AGM region from E10 to E12 were respectively separated by grinding gently, digestion by collagenase and filtering through the grit. The stromal cells of above region were obtained by discrepancy adherence, and was observed under a micorcsope.[Results] The cadiocyte and the superfic endotheliocyte of E10 to E12 both expressed the CD34 antigen (the surface marker of blood vessel), with the highest expression at E11. Meanwhile the hematopoietic cells in the circulation also expressed the CD34 protein. Jagged land Notch1 protein was expressed on all stromal cells in the spherical netword strutere of the heart, the former higher than the latter. Their expressions were strongest at E10.5, and gradually decreased as the embryo developed until E12. The vascular endothelial cells expressed the Jagged1 protein, but not Notch1. Notch1 protein was dispersed homogeneously in the surface of c-kit+ cells separated by immunomagnetic beads. The adherent cells from the embryo heart of E10～E12 were constituted by the difform cadiocyte with affluent kytoplasm and the slender endotheliocyte, which arranged in paralleled regularly and was seen easily to stick on the cadiocytes. CD31 antigen was expressed in the adherent of the heart, but not in the stromal cell of fetal liver from E10～E12, which showed uniform morphous with affluent endochylema. The stromal cells from AGM region were similar to the adherent cells of the heart.[Conclusion] The cadiocytes and the endotheliocyte from the embryo heart of E10 to E12 both expressed the CD34 antigen, indicating they had the characteristics of vascular endothelial cells. And the network structure constituted by the cadiocytes and the endotheliocyte with high expression of Jagged1 and Notch1 provided the favourable microenvironment for embryo hematopoiesis, inferring that the heart could participate in the development and amplification of HSCs.PartⅢ[Objective] To investigate the expression level of Notch signal pathway in the hematopoietic related region and explore preliminarily the position for HSCs' expansion during E10 to E12.[Methods] The gene expression of Notch1 and Jagged1 were respectively detected by real-time PCR in the stromal cells from the heart and fetal liver during E10 to E12 continuously. The rate of c-kit+ cells was monitored the BD-LSR flow cytometer from E10 to E12. The suspension cells of the heart at E11 were respectively cultured in co-culture system based in the stromal cells from the heart, the fetal liver and AGM region with the same conceptus age, and c-kit+ cells were analyzed by flow cytometer, estimating the effect of different stromal cells on HSCs' amplification.[Results] The gene expression level of Jagged1 and Notch1 was unchanged between primary cells and passage cells of adherent cells from the embryo heart, despite the passage made some difference to the morphous of adherent cells. Compared to the E10, the Jagged1's expression of E10.5 reached the maximum with 5.46±2.2 fold, but decreased at E11 with the same to E10. The Jagged1 's level of adherent cells from the heart at E11.5 and E12 were very low with 0.15±0.44 and 0.12±0.50 respectively to E10. The Jagged1 gene level of stromal cells from fetal liver during E10～E12 were low level except for E10. The rate of c-kit+ cells in the heart increased gradually from E10 with the highest level of 12.6±3.2% at E11, which was nearly similar to the fetal liver, except for E12 when the c-kit+ cells was 3.4±1.2% in the heart compared to the fetal liver with 11.6±4.1% c-kit+ cells. The rate of c-kit cells in the suspension cells from the heart at E11 maintained stable after 24h culture based in the stromal cells of the heart and AGM region with the same conceptus age, but the rate of c-kit cells obviously decreased in the condition of the stromal cells from the fetal liver. The c-kit cells of the three conditions were all at low level at 48h without significant difference.[Conclusion] The expression level of Jagged1 gene in the adherent cells from the heart was changed during E10 to E12, and reached the highest level at E10.5, with the expansion of HSCs concomitantly. Notch signal pathway may not play the important role in HSCs development in the fetal liver. It was inferred that the embryo heart and AGM region of E11 may contribute to HSCs amplification.