Transcription Factor RBP-J, Via Notch Signaling Regulates Cell Differentiation In Neural Development

The development of the Central nervous system (CNS) is one of the most complicatedly-regulated and highly-ordered process in the embryogenesis in vertebrates. The neural stem cells (NSCs) in the neural tube is a type of multipotent stem cell which while keeping self-renewal, can differentiate into different neural cell types. During neural development, NSCs change their intrinsic potency and response ability to extrinsic signals. At early developmental stages, NSCs experience accelerated proliferation to establish an enlarged progenitor pool. At later developmental stages, on the induction of various neurogenic signals, NSCs express proneural genes, such as bHLH family genes Mash1, Ngn1/2 and Math1, which initiate the expression of neuronal specific genes and promote NSCs differentiate into different types of neurons. On the initiation of bHLH genes expression, the homogeneous NSCs in the neural tube become heterogeneous, with proneural genes positive NSCs differentiate into neuronal progenitors, while those proneural genes negative NSCs maintain the stem cell property. The neuronal progenitor cells then exist the cell cycle, migrate into the mantle zone of the neural tube and terminally differentiate into neurons. On the other hand, the NSCs continue to proliferate, and produce late-generated neural cell types, such as glial cells, on responses to inductive neurogenic signals during late developmental stages. The maintainance of the neural progenitor pool is pivotal for the normal development of the CNS. The precocious differentiation and the insufficiency of NSCs proliferation results in the decrease of overall neural cells. Meanwhile, the precocious differentiation of NSCs only produce the early-generated neural cell types, leading to the loss of late-generated neural cell types, therefore completely impeded the normal formation of the CNS.The retina is an excellent model for research on the proliferation and differentiation of NSC/NPCs. During mammalian development, the retina progenitor cells (RPCs) give rise to seven retinal cell types, which are gradually generated in a conserved chronological sequence: ganglion cells and horizontal cells are born first, followed by cone photoreceptors and amacrine cells during the middle stage of retinogenesis. Rod photoreceptors, bipolar cells and Müller glial cells are the last cell types to be generated, mainly during postnatal stages. In addition to adopting specific cell fates, differentiating retinal cells need to migrate into appropriate laminae during retinogenesis, and thus, are eventually organized into three nuclear layers and two synaptic layers, based on which the light photons can be transduced into neural stimuli, and transmitted into the brain during the process of vision.Similar to the retina, the cerebellum, especially the cerebellar cortex, is one of the central regions in which ordered organizational patterns are most obvious. Compared with the cerebral cortex, its apparent simplicity and geometrical disposition have made it another ideal model for providing an understanding of the mechanisms involved in the development of the nervous system. During development, eight types of neurons and one type of glial cells are generated from the cerebellar ventricular zone and the rhombic lip, including Purkinje cells, Golgi cells, granule cells, basket cells, stellate cells, Lugaro cells, unipolar brush cells, candelabrum cells and Bergmann glial cells. During development, these cells are arranged into a stereotyped three-dimensional geometry, forming complex and accurate neural circuits responsible for important physiologic functions such as motor-coordinating and skill-learning.In neural development, the proliferation and differentiation of NSC/NPCs are regulated by many signaling pathways. Among them, the Notch signaling pathway composed of Notch receptors, transcription factor RBP-J and its downstream effectors, is one of the fundamental regulatory pathways during development. Although previous work has revealed that Notch signaling play important roles in neural proliferation and differentiation, various manipulations of different Notch signaling molecules yielded inconsistent results, and also raised different candidates of downstream genes. These might be attributed to that preceded studies have focused on the roles of single Notch receptor or the functions of individual downstream effector genes, which impeded the uncovering of the full scope of Notch functions in retinogenesis. In mammals, Rbpj encodes the transcription factor that integrates signals from all four Notch receptors and mediates the transcriptional activation of Notch target genes. Therefore, in the present study, we used the Cre-LoxP system to conditionally knock out Rbpj in the mouse RPCs and the NPCs in the cerebellar primordium.Rbpj-deficient retinae exhibited severe developmental disorders accompanied by reduced eye size, compared with that in wild-type animals. In the early retinogenesis, RPCs differentiation was accelerated in the absence of Rbpj expression, as reflected by the enhanced expression of proneural genes for all retinal neurons compared with the controls. However, only photoreceptors and ganglion cells were overrepresented, and other neuronal populations were diminished in the later retinogenesis. These results suggest that RBP-J deficiency led to the precocious differentiation of early born neurons and the depletion of RPCs. In the late retinogenesis, postnatal deletion of Rbpj in retina at P0, P3 and P5 led to increased photoreceptor production and decreased gliogenesis, indicating that RBP-J regulates the cell fate specification of photoreceptors and Müller glial cells. In addition, the retinal laminar structure was distorted accompanied with the gaps in the expression ofβ-catenin, the cell adhension-associate molecule, at the apical surface of the retina. And interestingly, lamination defects in Rbpj-deficient retinae were rescued by the induced-overexpression ofβ-catenin using in vivo electroporation.Rbpj-deficient cerebellum were severely reduced in size, and lost the folial structure compared with the wild-type cerebellum. At early developmental stages, the expression of proneural gene Mash1 was upregulated in the cerebellar ventricular zone, with neurons prematurely differentiated and NPCs significantly reduced. These results suggests that Notch-RBP-J signaling maintains the NPCs in cerebellar primordium and regulates the timing of cerebellar neurogenesis. At late developmental stages, Purkinje cells and granule cells were greatly reduced and Tbr1 positive deep cerebellar nuclei were almost lost. In addition, the transcription factors and secreted factors of the isthmic organizer (IO) were all expressed in the midbrain hindbrain boundary of the Rbpj-deficient embryos, indicating that the inactivation of Rbpj expression have no obvious effect on the formation of IO. These results also elucidate that Notch-RBP-J signaling directly involved in cerebellar development, instead of regulating cerebellar neurogenesis via affecting IO formation.In summary, our data indicate that RBP-J and, by extension, canonical Notch signaling inhibit neuronal cell type differentiation in neurogenesis, and therefore maintain the amount of NSC/NPCs till late developmental stages. In addition, Notch-RBP-J signaling participates in retinal cell specification, such as inhibiting photoreceptor production and promoting Müller glial cell differentiation in the retina developmental context. On the other hand, Notch-RBP-J signaling is likely to be involved in morphogenesis as well during neural development. RBP-J participates in retinal lamination through regulating the expression of cell adhesion molecules, such asβ-catenin. Meanwhile, our results also indicate that RBP-J dose not affect the pattern formation of midbrain hindbrain boundary. Taken together, our research demonstrate that transcription factor RBP-J is essential as a multifunctional regulator in NSC/NPCs specification and differentiation; In addition, RBP-J is also involved in morphogenesis in specific developmental context, therefore coordinates neurogenesis and morphogenesis, and ensures the normal establishment of the structure and function of the CNS. In that way, our research work has offered theoretical and practical significances in the understanding of the mechanisms of neural development, the uncovering of the full-scale Notch function, and the elucidating of the pathological mechanisms underlying related diseases of the CNS.