| Neural tube defects(NTDs)are the most common and serious developmental defect of central nervous system in clinic,which bring heavy burden to society and family.Neural tube defects are the disease caused by abnormal early embryonic neural development,which is a congenital nervous system developmental disease caused by closure disorder from neural plate appearance to neural tube closure.It is a serious central nervous system malformation disease,whose main manifestations are anencephaly,encephalocele,meningocele,spina bifida/recessive spina bifida,cleft lip and cleft palate.Neural tube developmental defects are usually caused by genetic,environmental and maternal factors,and its pathogenic mechanism is very complex.As a consequence,an in-depth study of the mechanism of neural tube defects is a key scientific issue in early embryonic development,pediatrics and other disciplines.It has important scientific and clinical significance for the prevention and treatment of diseases.The development of neural tube in early embryo mainly includes the following two important processes:neural induction and self-renewal of neural stem cells(NSCs).Neural induction refers to the process in which the notochord mesoderm induces the ectoderm to develop into a neural plate and then closes to form a neural tube during the early embryonic development.In addition,the NSCs self-renewal after the formation of the neural tube is the cytological basis of the early development of the neural tube.As a consequence,the study on the regulation mechanism of neural induction and NSCs self-renewal is the key to reveal the pathogenic mechanism of NTDs.The PcG protein family was first found in Drosophila melanogaster.It is considered to be a class of proteins that inhibit target genes and stably inhibit transcription by regulating the epigenetics of histones.PcG protein includes two complexes,PRC1 and PRC2.PRC1 plays an important role in the regulation of early embryonic development.It can regulate the differentiation of embryonic stem cells and is also related to the differentiation of the three germ layer.There is a protein in the PRC1 complex that can form a dimer with RING1A/B,called Pcgf protein(including six subtypes).Pcgf protein is very important in embryonic development.Pcgf1 plays an important role in the early brain development of embryo.It is mainly expressed in the nervous system.In vitro studies,it was found that there are serious defects in the differentiation of ectoderm and mesoderm of embryonic stem cells without Pcgf1.It was found that Pcgf1 maintains the pluripotency of P19 cells by regulating Oct4.The growth of zebrafish decreases in the early stage of development after the absence of Pcgf1,and about 35%of the fish with Pcgf1 deficiency mature prematurely.However,these studies have not proved the role and mechanism of Pcgf1 in early neural tube development.Taking zebrafish as the model animal and using Morpholinos to inhibit the expression of Pcgf1,we found that zebrafish embryos showed obvious neural tube development defects:delayed development,telencephalon decrease or even loss,shortening of body axis and other phenotypes.The neural induction process was abnormally activated,then the ability of NSCs proliferation decreased and cells drop out of the cell cycle ahead of time,resulting in a decrease in the number of NSCs and damage to neural tube development.In vitro,we used P19 cells to simulate the neural induction process and found that knock down of Pcgf1 caused P19 cells to enter the neural induction stage ahead of time,and the expression of pluripotent genes decreased.In our study we confirmed the important role of Pcgf1 in neural tube developmental defects and its regulation in neural induction and NSCs self-renewal in vitro and in vivo.In addition,we explored the dynamic regulation of early development-related transcription factors by Pcgf1 through epigenetic modification.Besides,we established a regulatory network of epigenetic factors and transcription factors in early neural development.Part ?:The expression pattern and influence of Pcgf1 during early neural tube development in embryo.We analyzed the spatio-temporal expression of Pcgf1 in zebrafish embryos by using RT-PCR?qPCR and in situ hybridization.The results showed that mRNA of Pcgf expressed differently at different developmental stages.Pcgf1 is maternally expressed in the embryonic development stage of zebrafish.Its expression increases from the sphere stage and increases significantly at the shield stage.In the shield stage,the development of the embryo enters the gastrula stage and begins to differentiate into the three germ layers,which is the key period of neural induction,indicating that Pcgf1 is specific in early embryonic neural development,especially in the neural induction stage.The results of in situ hybridization showed that Pcgf1 was specifically expressed at 16hpf stage,mainly concentrated in the central nervous system.In addition,it was expressed in the whole neural tube region,especially in the head and tail.The expression of Pcgf1 was significantly increased at the junction of forebrain,mesencephalon and telencephalon at 24hpf.We used MO to knock down the expression of Pcgf1 in zebrafish embryos.The phenotype of zebrafish embryos showed that knockdown of Pcgf1 led to obvious abnormal telencephalon development of embryos at 16hpf and 24hpf,such as microcephaly,reduction or even loss of telencephalon,and shortening of body axis.Part ?:The role of Pcgf1 in neural induction and self-renewal of neural stem cells.We analyzed the expression of several neural markers in neural induction stages(ie.shield,75%-epiboly phase and 10hpf)in order to further demonstrate the relationship between telencephalic defects caused by Pcgf1 knockdown and neural induction.The results of in situ hybridization showed that neural markers Sox2,Otx2 and Ngn1 were abnormally activated,but the expression of Sox3 increased at shield stage and decreased from 75%epiboly after Pcgf1 knockdown.In the meanwhile,qPCR further demonstrated that knockdown of Pcgf1 could lead to abnormal activation of neural induction.At the same time,we used P19 cells to carry out experiments in vitro.P19 cells can differentiate into neuroectoderm after RA inducing which can be used to study the process of neural induction.First of all,we found that the expression of Pcgf1 increased after the increased expression of neural markers Pou3f1 and Zfp521.However,the expression of pluripotent markers Oct4 and Nanog decreased.We constructed a stable P19 cell line with Pcgf1 knockdown and overexpression in order to explore the role of Pcgf1 in cellular pluripotency.It was found that P19 cells showed morphological changes after knocking down Pcgf1.Compared with the Ctrl group,P19 cells gathered earlier and entered the stage of neural induction in advance.In addition,we examined the effect of Pcgf1 on pluripotency and neural marker genes.qPCR and Western blot analysis showed that the expression of neural markers Pax6,Pou3f1 and Zfp521 increased at 24h and 48h after RA induction.At the same time,pluripotent markers Oct4,Hes1 and Nanog decreased after knocking down Pcgf1.Overexpression of Pcgf1 decreased the expression of neural marker Pax6 at 24h and 48h after RA induction,and increased the expression of pluripotent marker Nanog at 48h.Western blot results showed that the expression of pluripotent marker Oct4 was increased.This suggests that Pcgf1 may maintain the pluripotency of cells.In addition,the proliferation,apoptosis,differentiation and migration of neural stem cells are also the cytological basis for the normal development of neural tube.We do not know that the premature activation of neural induction caused by the knockdown of Pcgf1 will also affect the self-renewal of NSCs.First of all,we analyzed the expression of several neural markers in the self-renewal stage of neural stem cells(16hpf and 24hpf).The results of in situ hybridization and qPCR showed that the expression of neural markers decreased significantly after knocking down Pcgf1.Moreover,the results of BrdU and in situ hybridization of PCNA also showed that the levels of BrdU positive cells and the expression of PCNA were significantly decreased in the Pcgf1 MO group.Meanwhile,Acridine Orange staining also detected the apoptosis of neural stem cells.The results showed that the apoptosis of neural stem cells was not significantly affected.CDK inhibitors are important for differentiation.In addition,it was found that the expression of P21 and P57 increased in the group injected with MO compared with the Ctrl group.These results suggest that the abnormal development of neural tube may be caused by abnormal neural induction after Pcgf1 knockdown and subsequent abnormal proliferation and premature differentiation of neural stem cells.Part ?:The study on the mechanism of Pcgf1 in the process of neural induction and self-renewal of neural stem cells.We used RNA-seq technique to detect the changes of gene expression during neural induction of Ctrl group and Pcgf1 knockdown P19 cells in vitro in order to explore the mechanism of Pcgf1 on neural induction.The expression of 1745 genes changed more than twofold.After Pcgfl knockdown,the expression of 1502 genes were up-regulated and the expression of 243 genes were down-regulated.We also identified the types of significantly changed genes by GO analysis.These genes are associated with developmental phenotypes,such as mesoderm development and nervous system development.GO analysis also found that the down-regulated genes mainly included key transcription factors in the neural induction signal pathway,such as BMP and Wnt signal pathways.The expression of pluripotent genes(Nanog and Pou5f1)was also down-regulated after knocking down Pcgf1,indicating that pluripotency decreased and neural induction was advanced.In the meanwhile,the expression of histone demethylase was significantly up-regulated,indicating that Pcgf1 may also regulate neural induction through epigenetic mechanism.The regulation of neural induction process also includes the coordinated regulation of a variety of signal pathways in zebrafish,such as Wnt,BMP pathways and so on.GO analysis shows that these two signal pathways may be affected by Pcgf1.Our results showed that the activity of the transcription factor of BMP signal pathway(Smad1,Smad4,Smad5)decreased after Pcgf1 knockdown,especially Smad4,but the key regulators of Wnt signal pathway(Wnt3a,Wnt8a,?-catenin)did not change significantly.However,injecting the Smad4 mRNA after knocking down Pcgf1 did not significantly save the embryonic phenotype of zebrafish.It suggests that there may be other mechanisms that may affect the regulation of neural induction by Pcgf1.PcG gene plays an important role in the process of development.Epigenetic deformation of chromatin can lead to activation and silencing of specific genes.We found that both H3K4me3 and H3K27me3 decreased after Pcgf1 knocking down.ChIP-qPCR results showed that the level of H3K27me3 on Ngn1,Otx2 promoter decreased after the injection of Pcgf1 MO.In addition,the level of H3K4me3 on Pou5f3 and Nanog promoter also decreased.Taking these all together,Pcgf1 may lead to transcriptional activation and also transcriptional inhibition.Pcgf1 may regulate the process of neural induction through signal pathways and epigenetic.ConclusionZebrafish embryos show the phenotype of microcephaly and even telencephalon loss after Pcgf1 knockdown.Moreover,the neural induction process of zebrafish embryos is also abnormal.Subsequently,the self-renewal of neural stem cells is also inhibited after Pcgf1 knockdown.In the meanwhile,the results of P19 cells in vitro also showed that knockdown of Pcgf1 would cause P19 cells to enter the neural induction stage ahead of time,and the pluripotency of P19 cells decreased.It is found that Pcgf1 regulates nervous development through both signal pathways and epigenetic mechanisms through the analysis of key transcription factors of neural induction-related signal pathways and epigenetic.PcG protein is not only a transcriptional inhibitor but also a transcriptional activator.Pcgf1 regulates neural induction through inhibiting the expression of neural development-related genes through H3K27me3 and promoting the expression of pluripotent genes through H3K4me3.Innovation:1.This project first reveals the role of Pcgf1,a component of important epigenetic factor PRC1,in early neural development and neural malformation in vivo.2.We explore the molecular mechanism of Pcgf1 in regulating embryonic neurogenesis.3.It is proved for the first time that Pcgf1 regulates neural tube development through histone methylation in the early stage of neural induction. |
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