Regulatory Roles Of IL-3 In Development And Differentiation Of Hemangioblast During Mouse Embryogenesis

The cellular origin of hematopoietic lineages is the hot point in the field of developmental hematopoiesis and remains poorly understood up to date. Hemangioblast is an important model hypothesized 80 years ago, representing a common progenitor cell of hematopoietic and endothelial lineages. BL-CFC is the first direct evidence, and the model has been applied extensively to investigate the regulatory mechanisms underlying development and differentiation of hemangioblast from embryonic stem cells.. Recently, The HPP-HA model implies the existence of hemangioblast in AGM region. In this study, we aim to establish the BL-CFC model in AGM region to probe the modulatory pathway of hemangioblast activity during definitive hematopoiesis.As a first step, we set up the BL-CFC model in the AGM region. The cells of AGM region could form the typical blast colonies in the presence of bFGF, SCF, VEGF, IL-6, IGF-I and LIF for five days in semisolid culture medium. An individual colony could produce both nonadherent hematopoietic and adherent endothelial-like cells in vitro upon induced under specific conditions. As expected, the cells from an individual blast colony were capable of forming various kinds of hematopoietic colonies, including CFU-E, CFU-GM, CFU-Mix and CFU-HPP. Furthermore, the adherent population could incorporate LDL, was positive for CD31, vWF and SMA, but negative for CD45 or F4/80. Functionally, the adherent cells could form tube-like and network structures in Matrigel in vitro, and develop into capillaries-like structures in Matrigel plug in vivo, suggesting that they were authentic endothelial cells. Almost all the BL-CFCs were located in the AoM other than GM, and were exclusively enriched by CD31 or Tie2. Finally, the clonal nature of the bi-potential precursors was validated by a cell mixing experiment, in which AGM cells of wild type male (Sry+) and GFP+ female (Sry-)mice were cocultured initially and blast clolonies were individually collected for PCR to determine their genome DNA. Likewise, umbilical arteries contained the BL-CFC. Take together, these data suggested that the AGM-derived BL-CFC, like that from embryonic stem cells, is a reliable model of intra-embryonic hemangioblast.Based on the novel hemangioblast model, we screened multiple cytokines, including IL-3, bFGF, IL-6, Epo, TPO, GM-CSF, SCF,OSM, VEGF,for a preliminary appreciation of their effects on hemangioblast. We found that only IL-3 and bFGF can dramatically amplify the BL-CFC pool of AGM region. In addition, the hematopoietic and endothelial commitment of the BL-CFC were significantly enhanced by IL-3 incubation. The expansion effect of BL-CFC is IL-3 dose-dependent and could be abolished by IL-3 neutralizing antibody. Furthermore, IL-3 could promote proliferation but inhibit apoptosis of AGM cells. IL-3 mediated amplification was significantly reduced by supplementing AG490 or PD98059, suggesting a critical involvement of JAK-STAT and MAPK signaling pathway. Quantitative PCR demonstrated that IL-3 treatment lead to transcriptional changes of several hematopoietic- and vascular-related genes.In conclusion, we have provided unambiguous data showing that hemangioblast, the BL-CFC, emerges in the AGM region, and the novel model will be valuable in dissecting molecular mechanisms underlying the hemangioblast development and differentiation in the future.