The Complicated Network Structure and Activated Notch Signal Pathway in Embryo Heart of Mouse Provide the Favorable Microenvironment for HSCs Development and Proliferation During E10∼E12

Abstract 4806

Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process which (that) involves multiple anatomical sites. Although the AGM region is the origin of definitive haemopoiesis, it is known a little about its anatomical structure and the microenviroment in vivo (Are you using mouse for in vivo experimets?). Also it is little reported about whether there is other location for HSCs development and sharp amplification during the appearance of AGM region (from E9.5 to E10.5). Mounting numbers of documents indicated that AGM region was neither able to contain a great quantity of HSCs nor support the differentiation of HSCs. Through Sagittal slices of mouse embryos we found that the fetal heart presented the globular appearance with cancellous spatial structure at E9.5, constituted by the slender endotheliocytes linking to each other, expressing highly Jagged-1 ligand and Notch1 receptor. It contained many cycloid mononuclear cells with abundant cytoplasm and round nucleus, similar to the cells in blood vessels, with characteristics of hematopoietic progenitor cells expressing CD34. As the embryo developed, the shape of the heart changed quickly, with network structure diminished gradually. And at E12 the heart became as the hollow organ, sharing (owning) the characteristic of adult heart. Through immunohistochemistry the expression of Jagged1 reached the peak in fetal cardiac endotheliocytes of E10.5, and with the embryo development and morphological changes of the heart, Jagged1 expression level gradually decreased, and almost disappeared at E12. The expression of Jagged1 gene in cardiac endothelial cells of E10.5 was found in the highest level on mRNA level, when compared to E10's (5.46 ± 2.2) times. Jagged1 expression in E11 decreased, almost as the same as in E10, and Jagged1 expression of cardiac endothelial cells in E11.5 and E12 were very low, respectively, of E10 (0.15 ± 0.44) times and (0.12 ± 0.50) times. However, Jagged1 gene expression in fetal liver stromal cells in E11 was similar to the cardiac endothelial cells in E10, the rest showed at low levels of expression. By flow cytometry analysis we found that the c-kit+ cells in the embryonic heart and fetal liver of E11 were almost identical, when the c-kit+ cells increased significantly, respectively (12.6 ± 3.2) % and (9.6 ± 2.8) %. But the c-kit+ cells in the heart of E12 decreased significantly, only as (3.4 ± 1.2) %, when c-kit+ cells in fetal liver were (11.6 ± 4.1) %. We found that the suspension cells of E11 were co-culture with cardiac endothelial cells and AGM stromal cells for 24h, c-kit+ cells remained stable, but was significantly reduced in co-culture with the fetal liver stromal cells. In co-cultured for 48h, three groups of c-kit+ cells showed a low level, although there was no significant group difference. We speculate that the greatest surface area in embryo heart constituted by the complicated network structure and activated Notch signal pathway could provide the favorable microenvironment for HSCs development and proliferation utmostly during E10∼E12.