Identification Of Cell Surface Markers And Transcriptional Regulation On Development Of Neural Stem Cells

Neural stem cells have the potential to produce different types of neural cells through self-renewal and differentiation,and are the cellular basis for the complex structure and function of the brain.As development proceeds,the neural stem cell population becomes hetergenous,and the number of neural stem cells in the brain gradually decreases.The morphology,cell biological characteristics,and the progeny cell types produced also vary correspondingly.Because of lack of specific molecular markers,neural stem cells and progenitor cells can not be clearly defined at the molecular level.Therefore,identification of surface markers for isolating and purifying different subtypes of neural stem and progenitor cells is of great significance to understand neural stem cell biology and expand its application in translational medicine.During cerebral cortical development,neural stem cells produce different cell types in a highly ordered manner,which is precisely regulated by an intrinsic transcriptional regulatory network.Dissecting the transcriptional regulatory network at different stages of neural stem cell development will help understand the mechanisms underlying neural stem cell fate determination,and provide new strategies for in vitro differentiation and treatment of neurological diseases.In this dissertation,I used mice as model animals to investigate these two aspects of neural stem cell development in the embryonic cerebral cortex.To identify surface markers of neural stem cells,we collaborated with chemists to develop a metabolic glycan labeling method using un-natural sialic acid metabolic markers.Utilizing a neural stem cell-endothelial cell co-culture system to amplify primary neural stem cells isolated from mouse embryonic cortex in vitro,we were able to isolate membrane proteins from neural stem and progenitor cells for Mass-spectrometry analysis.We have identified a list of membrane proteins that are specifically expressed or enriched in neural stem and progenitor cells,compared to differentiating cells.We have discovered that Igsf8 is highly expressed in intermediate progenitor cells and is expected to be the first surface marker for isolating and purifying this type of neural progenitor cells,which plays an important role in cortical neurogenesis.In order to study the molecular mechanisms involved in gene transcription regulation of neural stem cells during the development of embryonic cerebral cortex,we focused on FOXG1,a transcription factor specifically expressed in the forebrain,as a starting point.We demonstrated the expression pattern of FOXG1 in the developing cerebral cortex,and performed chromatin-immunoprecipitation followed by sequencing analysis on FACS-purified neural stem and progenitor cells from different stages of development.We found that FOXG1 was dynamically distributed in the genome of neural stem and progenitor cells.In combination with bioinformatic analysis,we identified transcription factors that interact with FOXG1 in vitro.In summary,my thesis project has established a new method for efficient identification of surface markers for neural stem and progenitor cells,and identified Igsf8 as a putative surface marker for intermediate progenitor cells.This dissertation also describes the genome-wide dynamic distribution of the transcription factor FOXG1 in neural stem and progenitor cells,and the identification of transcription factors interacting with FOXG1.Our findings lay a foundation for further characterization of the different types of neural stem and progenitor cells and their functions,and the regulatory mechanisms underlying their fate determination,to ultimately help promote the application of neural stem cells in translational medicine.