title: Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region.
abstract: BACKGROUND
  The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0-7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined.
  
  
  METHODS
  The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity.
  
  
  RESULTS
  Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19(+) B lymphocytes, and CD3(+)TCRbeta(+) T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31(+) tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31(+), endomucin(+) and CD105(+) subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis.
  
  
  CONCLUSIONS
  The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.
authors.affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China.
authors.forename: Wen-Yan
authors.initials: WY
authors.lastname: He
authors.forename: Yu
authors.initials: Y
authors.lastname: Lan
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Yao
authors.forename: Zhuan
authors.initials: Z
authors.lastname: Li
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Wang
authors.forename: Xiu-Sen
authors.initials: XS
authors.lastname: Li
authors.forename: Ji-Yan
authors.initials: JY
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Ning
authors.initials: N
authors.lastname: Mao
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D006066:Gonads / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D055018:Hemangioblasts / Q000166:cytology / Q000502:physiology*; D006410:Hematopoiesis / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000502:physiology*; D008650:Mesonephros / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D051379:Mice; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology
chemical_list: D007377:Interleukin-3