Study Of The Embryonic Cortex Neurogenesis Process

Neural stem cells (NSCs) are self-renewal multipotent cells that generate neurons and glia cells during embryonic development. In the embryonic period, the cerebral cortex forms a six layers structure with different types of neurons with an"inside-to-outside" pattern through the process of continuous NSCs proliferation,neuronal differentiation and migration. Some neural stem cells persist in the adult vertebrate brain and continue to produce neurons throughout life. Stem cells are characterized by their capacity to differentiate into multiple cell types. They undergo symmetric or asymmetric cell division into two daughter cells. In symmetric cell division, both daughter cells are also stem cells. In asymmetric division, a stem cell produces one stem cell and one specialized cell.During embryonic development, neural stem cells are mainly located in the two regions of the cortical region, the VZ and SVZ. Radial glia cells (RG) in the lateral ventricle region maintain self-renewal by symmetric division and produce intermediate progenitor (IP) cells by asymmetric division. IP cells then produce neurons, which migrate to a specific area to form a complete cerebral cortex along the radial processes of RG cells. In this process, various transcription factors are activated continuously (Pax6-Tbr2-Tbrl-Cux1-Satb2). If any step in this process are interrupted or disturbed, it can lead to serious developmental disorders such as schizophrenia and autism. However, the in-depth mechanism of neural stem cell proliferation, differentiation and migration needs to be further studied.In the process of embryonic cortical development, various forms of epigenetic regulation (histone methylation, acetylation, phosphorylation; DNA methylation;microRNAs) play important roles. In these different epigenetic modifications, histone variants belong to a large family, including H2A.Z, H2A.X, macroH2A and H3.3.We focus on studying H3.3, a very critical histone variant. The nucleosomes of eukaryotes have different types of histones, including H1, H2A, H2B, H3 and H4.The composition of histones in nucleosomes undergoes dynamic changes during gene transcriptional activation or inhibition. At the time of transcriptional activation,H2A.Z and H3.3 are inserted into nucleosomes to form a chromosome activation complex that affects the accessibility of proteins and DNA at chromatin levels,thereby facilitating transcription. Histone variants play a regulatory role in the expression of genes at specific regions of the genome. Therefore, the study of transcriptional regulation by histone variants will contribute to a deeper understanding of gene expression mechanisms. At the same time, the introduction of histone variants into the study of early embryonic neural stem cells will give a new perspective to reveal their important role in neurodevelopment. Previous studies have found that H3.3 can replace H3, when the chromosome is in a transcriptional activity state. H3.3 contains a rich modification sites, and it will undergo different epigenetic modifications.In our experiment, we found that H3.3 was expressed in neural stem cells in vivo and in vitro. In utero electroporation (IUE) experiments showed that H3.3 knockdown led to decrease the proliferation of NSCs, and increase the proportion of neuronal differentiation. Through further studies, we found that H3.3 knockdown resulted in a decrease in H4K16ac. H3.3 knockdown caused the reduction of recruitment of H4K16-specific acetyltransferase, thereby reducing the level of H4K16ac, which affects the transcriptional activation of genes. All these results suggest that H3.3 and H4K16ac have the synergistic effect in the development of neural stem cells to regulate the process of embryonic neurogenesis. Through the transcriptome sequencing, we found that a large number of biological processes were affected when H3.3 was knocked down. We analyzed the data and found that the expression of all members of the GLI family were decreased, and GLI1 was the most reduced gene.We found that Glil was a downstream factor of H3.3.In the development of the cerebral cortex, glutamate is a necessary amino acid,which plays an important biological function. In mammals, glutamate receptors are divided into ionic glutamate receptors and metabotropic glutamate receptors.Metabotropic glutamate receptors are reported to be associated with neurological disorders. It regulates the release of neurotransmitters and neuronal excitability.Metabolic glutamate receptors are divided into three categories based on their protein sequences, functions and ligands. They are category I (GRM1 and GRM5), II(GRM2 and GRM3), and III (GRM4, GRM6, GRM7, and GRM8). It has been reported that GRM7 may be associated with ASD and ADHD. However, whether the absence of GRM7 affects the development of the cerebral cortex during the embryonic period is unknown.In our experiments, we studied the function of GRM7 in the development process by overexpressing and knockdown GRM7 in the cerebral cortex. The results showed that GRM7 was expressed in neural stem cells. GRM7 and neural stem cell markers were colocalized, and the expression of GRM7 in different developmental stages was dynamic. In vivo embryonic electroporation experiments showed that GRM7 knockdown led to increase NSCs proliferation and decrease neuronal differentiation.The morphological development of neurons is also affected. Mechanism studies showed that change of GRM7 expression affected the phosphorylation of CREB, and then p-CREB as a transcriptional regulator affects the expression of downstream genes. Subsequent experiments showed that YAP acted as downstream of GRM7 and p-CREB and plays a role in regulating the proliferation and differentiation of NSCs.