| Cyclin dependent kinase 5(CDK5) is a member of the cyclin-dependent-kinases family of serine/threonine kinases and was discovered in the early 1990s. CDK5 plays a multifunctional roles in the neurous system by virtue of phosphorylating diverse substrates. During development, it participates in neuronal proliferation, differentiation, migration, as well as axon outgrowth and synaptogenesis. Neurulation is an early developmental event that results in the formation of neural tube, the rudiment of the entire adult central nervous system(CNS). Recent studies of the developmental neurobiology have focused on the cellular and molecular mechanisms of neurulation. Findings from these studies reveal that neural stem cells are self-renewing, multipotent progenitors that give rise to the diverse types of neurons and glia. As to molecular and genomic level, the event of neural tube close involves the highly complex expression pattern and interactions of a series of genes. Neural tube defects (NTD) are one of the most common central nervous system malformation, regarded as a result caused by both environmental and genetic factors. In the recent studies, changes of the neural stem cell, extracellular matrix as well as migration signals, etc. were reported to be related with the occurrence of NTD. However, very little is known about the correlative genes and relevant pathways involving in the formation of embryonic neural tube and NTD. The roles and mechanisms of CDK5 involved in the neurogenesis, its regulative roles in the differentiation progression of neural stem cells remain largely unknown.In the present study, we established a model of retinoic acid (RA)-induced NTD through administrating RA (doses of 50mg/kg) to pregnant mice on gestational 7.5 days and harvested the normal and NTD embryo. The changes of distribution, number and cell cycle dynamics of neural stem cells during different developmental stage of the normal and the RA-treated embryo were examined using immunofluorescence on cells, whole-mount confocal of embryo by immunofluorescence labelling, semithin section staining and flow cytometry. The differentially expressed genes during the formation of embryonic neuraltube, as well as NTD, were analyzed by mean of middle-density DNA microarrary techniques. Finally, special attentions were paid to the expression changes of CDK5 during developmental brain and the possible roles of CDK5 in differentiation of neural stem cells, by using in situ hybridization (whole-mount embryo and section), immuneohistochemistry, cell culture and gene transfection methods. The main results were as follows: â… . The cell dynamic changes of the normal and the RA-treated neuroepithelium during embryonic development 1. We established the NTD model and showed that the central nervous system malformation at age of embryonic day 10.5(E10.5) included acrania, craniofacial abnormal, spina bifida, short-tailed or tailless ect. This indicated RA-treated embryo could generate various types malformation and the model was reliable.2. Using cells immunofluorescence labeling, flow cytometry and whole-mount embryoic confocal immunofluorescence technique, higher level of the nestin positive cells were detected in both fetal cerebral vesicle and spinal cord nerve tissue than that of adult brain in normal. It reached to peak at the 8th day of embryo and decreased gradually after E13.5d. We observed that the nestin positive cells distributed mainly near the internal limiting membrane and mantle layer of neuroepithelium. The positive cells arranged regularly along the neuroepithelial layer. After RA-treated, it was found that the number of neural stem cells reduced, especially at the eighth day of embryo (p<0.01). The nestin positive cells distributed disorderly. The results suggested that the occurrence of NTD may mainly due to the number of neural stem cells reduced during formation of the neural tube.3. We observed that there were many mitotic division cells in the ventricular zone and the subplate by using semithin section stainin...
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