| ObjectiveThe embryonic vasculogenesis and tumor angiogenesis possess similar correlations to some extent. PTEN has been regarded as one of the most important tumor suppressor gene. Therefore, We investigate PTEN role on hemopoiesis precursor cell migration and differentiation in early embryonic vasculogenesis, which hopefully could offer us basic theory support for the anti-tumor therapy in the future.MethodIn situ hybridization:Endogenous chick PTEN and VE-Cadherin expression pattern was firstly detected in early chick embryos using PTEN and VE-Cadherin RNA probe. Using the same strategy, we also analyses the gene expression pattern in over-expressed Wt PTEN.Labeling hemopoiesis precursor cells:The fertilized eggs were incubated until to HH3 embryos with EC culture, and then follow the migration of posterior primitive streak cells labeled with DiI or transplanted GFP-positive cells, which are the hemopoiesis precursor cells in the stage of primitive streak.Tracing hemopoiesis precursor cell migration:To investigate if PTEN gene is involved in modulating the vasculogenesis, we over-express Wt PTEN comparing the empty vector control pEGFP-N1 using electroporation after microinjection into the potential space between epiblast and vitelline membrane at gastrulation embryos, observe cell movement and analysis the GFP-positive cells contributing to blood islands after overnight continually incubation. In addition, we employed the similar strategy to the transplantation experiment in order to supply the experimental evidence, in which primary streak tissue transfected with pEGFP-Nl or Wt PTEN-GFP grafted into the same position in primitive streak of donor embryo, i.e. transplant the GFP/Wt PTEN positive primitive streak tissue to un-transfected donor embryos at chick embryonic stage HH3. In the next step, we try to analyze if over-expressed PTEN gene affect cells undergoing EMT in primitive streak or migrating/contributing to the position where blood island form. The advantage for transplantation experiment is of eliminating the interference of local mass GFP-positive cells and is more intuitive observation for cell migration trajectory.Identification for blood island with VE-Cadherin expression:Using VE-Cadherin in situ hybridization, the blood island formation is determined in the embryos of over-expressed PTEN genes. We check the change of morphology and function in phase of formation blood island in light of over-expression WtPTEN gene. Next, we use IPP and SPSS software to calculate and analysis blood island density in both of pEGFP-N1 and Wt PTEN-GFP groups.PI3K signaling inhibition:To block PI3K signaling pathway, we exposed PI3K inhibitor LY294002 in half of chick embryo while another half as control. After treatment of PI3K inhibitor the morphology and density of blood island will be analyzed as described above.ResultEndogenous chick PTEN gene begin to express in epiblast and mesoderm at chick gastrula stage, and then expression extend widely in embryonic area and area opaca (extra-embryo part i.e. vasculogenesis area later on). VE-Cadherin express in blood island of early chicken embryos, which are the common precursor cell for both vascular endothelial cells and hematopoietic stem cells. Using DiI labeling we trace posterior primary streak cell migration to area opaca, in which the precursor cells participate in vasculogenesis process. Transfected or allograft Wt PTEN-GFP in posterior primary streak resulted in epiblast cells restrained in EMT process comparing to the control pEGFP-Nl group, i.e. Wt PTEN-GFP positive cells mostly stick in the epiblast or within primitive streak. Although without influence on density of blood island by over-express PTEN gene in chick embryo (p>0.05), Wt PTEN-GFP positive cells were found to fail to involve in the formation of blood island, which is significant difference compared with PEGFP-N1 embryos (p< 0.01). Blocking PI3K signaling pathways by LY294002 generated little effect on blood island density (p>0.05), but lead to blood significant impact to blood island morphology, such as gathered clusters for most of blood island lost apparent shape for blood island and further primary vascular plexus.ConclusionPTEN gene is involved in the angioblast cell generation and migration originated from posterior primitive streak, i.e. angioblast undergoing EMT and movement to area opaca, in which blood island form (vasculogenesis). Meanwhile, the PTEN effect can't eliminate the possibility about indirect via PI3K signaling since morphology alteration was observed although no influence in blood island density after blocking PI3K signaling pathways. |
