engleski

COMPARISON OF EXPRESSION OF THY1 - YFP DURING EMBRYO DEVELOPMENT AND IN VITRO DIFFERENTIATION OF NEURAL STEM CELLS

Objective In order to analyze morphologic processes occurring during development of mammalian CNS, several specific mouse strains have been constructed. This study was based on the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J which under the control of Thy1 gene promoter expresses yellow fluorescent protein (YFP) in neurons. In this study, we have determined and compared pattern of Thy1 - YFP expression during embryonic development as well as during differentiation of neural stem cells culture. Material and methods Pregnant females, at different stages of gestation were used for embryo isolation. The embryos were fixed and cryosectioned on 20µm thick slices. Neural stem cells were isolated from the forebrain of 14.5 old murine embryos and cultured as neurospheres. After dissociation, cells were plated on cover slips. The cells were fixed 1, 3, 5 and 7 and prepared to the immunocytochemistry. Results First signs of expression were observed at embryonic day 12.5 in ventral horn of the spinal cord. During development of the embryo in the cranio-caudal direction. In accordance with the process of neurogenesis which primarily occurs in mid-gestation, expression of YFP in neurons after E12.5 became much more pronounced. With embryo maturation, expression became stronger in the brain and by E17.5 expression was observed in the optic nerve and retina. Furthermore, expression of YFP was determined during in vitro NSC differentiation. YFP expression was observed in neural progenitors as well as in mature neurons. YFP positive cells stained positive for Map2 (mature neuronal marker), while astrocytes which were GFAP positive were not expressing YFP. Conclusion Our results showed that, in addition to mature neurons, neuronal progenitor cells express this construct both in in vivo and in vitro conditions. This suggests that in addition to analyses of neuronal differentiation in B6.Cg- Tg(Thy1-YFP)16Jrs/J mouse, NSCs isolated from this strain can be successfully used in studies where tracing of cells with neuronal fate is needed.

stem cells; mouse; THY1-YFP; embryonic development

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