The Role And Mechanism Of Notch Signaling In Nsc Differentiation And Neuron Morphogenesis During Central Nervous System Development

Stem cells are captivating because they have the potential to make multiple cell types yet maintain their undifferentiated state (self-renew). Neural stem cells (NSCs) are the foundation of the nervous system. They are a small number of highly plastic cells that proliferate, acquire regional identities and differentiate into different cells (neurons and glial cells), and finally develop into the entire nervous system. These characteristics of the NSCs make them the most potent candidates for the therapy of neural degenerative diseases and injuries of central nervous system. But the application of NSCs into clinical therapy is not possible until we thoroughly understand the mechanisms that regulate the proliferation and differentiation of NSCs. Therefore, the study of the regulation of NSCs will not only improve our theretical knowledge about the neural development but also benefit the future NSC-based clinical therapy.So far, it is known that the proliferation and differentiation of NSCs are regulated by many factors including cell-autonomous ones and cell-nonautonomous ones. The signaling based on the cell membrane receptor named Notch is one of the most important factors that play an essential role in the regulation of NSCs behaviors. Notch signaling is critical for multiple aspects of neurogenesis, such as maintaining the undifferentiation state of NSCs, regulating the binary fate choice during NSC differentiation, and effecting the final maturation of progeny cells. But how it regulates the proliferation and differentiation of neural stem cells (NSCs) and intermediate neural progenitors (INPs) has not been well elucidated, especially in vivo.In this study, we are aimed to generate the transgenic mice that ablate the Notch signaling specifically in the central nervous system (CNS). Further, we would in vivo observe the phenotype of NSCs in this mouse line, to find whether the NSCs themselves have abnormality in their proliferation and differentiation, and to study whether the progeny cells produced by Notch-ablated NSCs show any defects. Finally, we hope to elucidate in vivo the multiple roles of Notch signaling during different stages of CNS development, and to investigate the underlying mechanisms.The results we have obtained are as follows:1?First, we successfully generated the NesCre transgenic mice which mainly express the Cre recombianse in the basal forebrain and ventral midbrain. Then we obtained the conditionally gene targeting mice that ablated the transcription factor RBP-J, which mediates signaling from all four mammalian Notch receptors, in CNS by mating the newly established NesCre mice with the RBP-J-floxed mice that we have successfully generated before.2?Second, we found the defects of NSCs in differentiation in the RBP-J-ablated mice. At early stage of neurogenesis (E11.5), the frequency of neurospheres increased significantly in the RBP-J-inactivated regions. But the majority of the RBP-J deficient neurospheres were composed of INPs, suggesting the precocious differentiation of NSCs into INPs. At late neurogenic stages (E17.5 and neonatal), as expected from precociously exhausted NSC pool, neurosphere frequency and NSCs decreased in the RBP-J-ablated regions, accompanied by a significant increase of both neurons and glial cells. Meanwhile, neuronal differentiation was reduced in the same regions at E11.5. These results indicated that the RBP-J-mediated signaling might inhibit the differentiation of NSCs into INPs and support the generation of certain early born neurons at early neurogenic stages.3?Third, neurons derived from RBP-J-deleted NSCs had some changes in morphology. When we ablated the Notch signaling in NSCs, not only the NSCs preferentially differentiated into neurons, but also the produced neurons showed to have more branches after further quantification.4?Finally, we investigated the mechanisms by which Notch signaling regulating the differentiation of NSCs and final morphology development of neurons. The microRNA chip result indicated several possible molecules that would play crucial roles in the regulation of NSC differentiation. And we found that microRNA342-5p could promote the branching of neurons during their maturation. Notch signaling could inhibit the expression of microRNA342-5p, and thus regulated the morphology development of neurons.In summary, our research elucidated the role of classical Notch signaling in the differentiation of NSCs into INPs, and indicated that microRNA molecules might involved in the regulation. Our results showed that Notch signaling, through regulating the expression of microRNA342-5p, regulated the final maturation of neurons. These research help us to understand the development of CNS, and the study about the relationship between Notch signaling and microRNAs further supply new ideas to the knowledge of mechanisms by which Notch signaling regulate the CNS development. These data also have shed light on the future clinical application of Notch signaling as potential therapy target.