Research Upon The Regulatory Mechanism Of Notch Signaling Pathway During The Differentiation Of Neural Stem Cells

The neural system is the most important and complicated system of our body. It results from neural tube and neural crest. Neural tube initially is composed of a flat of neuroepithelium. With neuroepithelial cells' large proliferation, differentiation and transplantation, three structures together form neural tube. There are many neural stem cells (NSCs) in neuroepithelium, which are self-renewing, multipotent progenitors that give rise to the diverse type of neurons and glia. Following the neural tube closure, neuroepithelium continued to proliferate, differentiate and migrate, and prosencephalon, mesencephalons and rhombencephalon appeared in the anterior region of neural tube. The proliferation, differentiation and migration of the neuroepithelium are essential for the structural formation and functional establishment of the nervous system. Neural tube defects (NTDs), resulting from failure of the neural tube to close properly during the early development of embryogenesis, are the most common severely disabling birth defects over the world. Nowadays, the studies of neuroepithelium development have focused on the study of associated candidate genes and the cellular and molecular mechanisms. Findings from these studies reveal that a number of genes participated in the regulation and control of development of the central nervous system and some environmental factors exert their functions via these genes as well.Among all the regulatory mechanisms, Notch signaling pathway has an intimate relationship with the development of neural tube, and its "lateral inhibition" has been thought to play a key role in the differentiation of NSC. All-trans retinoic acid (ATRA) is the normal metabolite of Vitamin A, which is a strong differentiation inducer, and it has an important function in the form of neural tube. ATRA-induced NTDs model have been widely used in the scientific researches, but the mechanism has not been reported since now. As the proliferation and differentiation of NSC have an important meaning to the form of neural tube. So, in the present study, we used Western blot, Real-time PCR, Dual-luciferase Reporter System, NSC culture, plasmids co-transfection and immunocytochemistry to investigate the influence of ATRA upon the Notch signaling pathway. This has established a theory basis and experimental evidence for later study on the mechanisms during the embryonic central nervous system development, especially the proliferation or differentiation of neuroepithelial cells and the genesis of NTDs. ChapterⅠThe Expression of Notch-Signaling-Pathway-related Genes during the Differentiation of Neural Stem CellsObjectiveTo establish procedures of neural stem cells cultivation and discuss their characters of proliferation and differentiation in vitro, and to investigate the influences of ATRA upon Notch-signaling-pathway-related genes (Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1 and Neurogenin 2) during the differentiation of NSCs.MethodsNeural stem cells were separated from the cerebral cortex in C57BL/6 mouse embryos (E15) mechanically and were cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. The culture conditions of neural stem cells were explored and the passaging methods were investigated. Then, all neural spheres were attached to the bottom and were induced to differentiate. The indirect immunofluorescence cytochemistry was used to identify the neural stem cells and the differentiated cells with the markers of Nestin for neural stem cells, neurofilament (NF) for neurons, glial fibrillary acidic protein (GFAP) for astrocytes and Galactocerebroside (GALC) for oligodendrocytes.NSCs differentiated in control groups and experimental groups added by 1μmol/L ATRA. NSCs and differentiated cells were collected at 0 (NSCs), 1, 3, 5, 7 days. Notch 1, Musashi 1, Numb, Presenilin 1, Rbpj, Hes 1, Sox 1, Mash 1 and Neurogenin 2 mRNA expressions were determined by Real-time PCR. The Notch intracellular domain (NICD) was determined by Western blot.ResultsPlenty of neural stem cells were obtained from the cerebral cortex by means of the mechanical dissociation and the serum-free medium. The obvious neural spheres can be observed in about 10 days and can be maintained in vitro for more than 8 weeks. The neural stem cells induced to differentiate in vitro can produce various types of neural cells. The indirect immunofluorescence cytochemistry showed Nestin positive for the neural stem cells and NF, GFAP and GALC positive for the differentiated cells.Compared with control groups, in the experimental groups, Notch 1 mRNA level and NICD were decreased, Musashi 1 mRNA level was increased, Numb mRNA level was increased at first then decreased later, Presenilin 1 mRNA level was increased, Rbpj mRNA level was decreased at first then increased later, Hes 1 mRNA level was decreased, Sox 1 mRNA level was decreased, Mash 1 mRNA level was decreased, Neurogenin 2 mRNA level was decreased.ConclusionsPlenty of neural stem cells can be obtained from the embryonic mice. These cells showed multipotential differentiation and can be induced to differentiate into various types of neural cells such as neurons and astrocytes.ATRA could advance NSCs's differentiation by inhibiting Notch 1 expression, which could be suggested by the depression of Notch 1, Hes 1 and NICD. The up-regulation of Musashi 1 and the later down-regulation of Numb accordingly may be related to some kind of regulatory mechanism of feedback due to the depression of NICD, and the up-regulation of Presenilin 1 and later up-regulation of Rbpj are related to this mechanism as well. The down-regulation of Sox 1 may be connected with the down-regulation of Hes 1. The down-regulation of Mash 1 and Neurogenin 2 suggested that NSCs differentiate into less neuron after the treatment of ATRA.ChapterⅡThe Function ofγ-secretase during the Differentiation of Neural Stem CellsObjectiveTo establish an assay, based on Gal4-VP16/UAS and Dual-luciferase reporter gene, for detecting the activity ofγ-secretase in the differentiation of neural stem cells. To detect the activity, enzymatic product (intracellular domain of Notch1, NICD) and catalytic subunit (Presenilin 1, PS1) ofγ-secretase for further studying the regulatory mechanism of NSCs' differentiation. To investigate the influence of ATRA upon the activity ofγ-secretase during the differentiation of NSCs.MethodsPlasmids Notch1△E-GVP, MH100 and pRL-CMV were cotransfected into NSCs, when they were under the condition of differentiation. Notch1△E-GVP encodes the transmembrane and intracellular domains of mouse Notch1, inserted with a Gal4-VP16 lacking an internal ATG after the transmembrane domain. MH100 encodes the upstream activation sequence (UAS) and Firefly luciferase. pRL-CMV encodes the Renilla luciferase. The activity ofγ-secretase was detected by Dual Luciferase Reporter Assay System. The productions of NICD were analyzed by Western blot. PS1 mRNA expressions were determined by Real-Time PCR. Resultsγ-secretase inhibitor DAPT inhibited the productions of NICD and it also decreased luciferase activities in a dose-dependent manner, while the PS1 mRNA level was increased accordingly. The activity ofγ-secretase was increased after the treatment of ATRA during the differentiation of NSCs.ConclusionsThe assay based on Gal4-VP16/UAS and Dual-luciferase reporter gene is effective and reliable for detecting the activity ofγ-secretase, which is helpful to the researches upon the proliferation and differentiation of NSCs. During the differentiation of NSCs, as for the regulatory range ofγ-secretase upon the Notch signaling pathway, the influence of ATRA upon the Notch signaling pathway is just due to its inhibitory effect on Notch 1 expression, instead of by suppressing the activityγ-secretase and the expression of its catalytic component Presenilin 1.