| Gestational diabetes is one of the most common complications during pregnancy,with serious impact on maternal and infant health,which can cause abnormal embryonic and neonatal neurodevelopment.The incidence of gestational diabetes is increasing year by year,with the change of the lifestyle and dietary habit,delayed marriage and reproductive age,and implement of new diagnostic criteria of gestational diabetes.Gestational diabetes seriously affects embryonic neurodevelopment.Hyperglycemia in pregnancy causes neurodevelopmental retardation and higher incidence of neuropsychiatric disease in offspring,in addition to cause birth defects,such as neural tube defects.Neurodevelopment disorder caused by maternal hyperglycemia is an important public health problem worldwide especially in developing countries.Maternal hyperglycemia disturbs neurodevelopment,which is an outcome of combined action of environmental factors and genetic factors,but its etiology and pathogenesis is still unclear.Therefore,it is a problem to be solved to reveal the pathogenesis that hyperglycemia in pregnancy causes abnormal neurodevelopment.As the most core structure of the brain,the cerebral cortex is an important center controlling cognition,behavior,motor and sensory.The development process of the cerebral neocortex includes the continuous expansion of neural precursor cells and ordered exit from the cell cycle,then successive differentiation generating neurons and glial cells.Experimental results show that hyperglycemia during pregnancy affects proliferation and differentiation of neural stem cells,suggesting that maternal hyperglycemia may disturb the embryonic corticogenesis.During forming process of the cerebral neocortex,the neural precursor cells differentiate into neurons first,then the newborn pyramidal neurons assemble from inside to outside.The early born neurons migrate outward first,forming the deep layer of neocortex(6 layer first,and then generate 5 layer).The later born neurons then migrate outward,forming the superficial layer(layer 2 to 4).It most likely to cause some neuropsychiatric disease,if the structure and neurocyte composition of the neocortex are disturbed,in result of that gestational hyperglycemia caused abnormal differentiation of neural stem cells in embryonic brain.Astrocytes also play an important role in synaptic formation and siagnal transmission.Abnormal astrogliogenesis also increase the incidence of neuropsychiatric disease.Epidemiological survey found that the offspring of gestational diabetes have a higher incidence of cognitive impairment and neurodevelopment disorder than the offspring of normal pregnancy in childhood and adult.But there are few researches on effect of maternal hyperglycemia on the development of cerebral neocortex,which mechanism also needs to be further discussed.In the process of formation of cerebral neocortex,epigenetic regulation plays an important role.There are complex and dynamic networks between histone modification and transcription factor,regulating the proliferation and differentiation of neural stem cells in the process of formation of cerebral neocortex.Activation of transcription factors related with neuronal differentiation by histone acetylation is main transcriptional modification in neural stem cell differentiation towards neurons.Research has shown that high glucose treatment dicrease acetylation level of histone H3K9 in neural stem cells under proliferative condition.Some studies indicate that the acetylation level of histone H3K9,K3K14,H3K27,H3K56 increase in the model of embryonic neural tube defects caused by gestational diabetes.But there is no research about the regulation of maternal hyperglycemia on the differentiation of neural stem cells in the process of the formation of cerebral neocortex.In this study,we examined the change of histone acetylation and transcriptional activation of proneural and neuronal factors,to explore the relationship network across epigenetic modifications and transcription factor regulating neural stem cell differentiation under the condition of high glucose.During the embryonic neurodevelopment,microRNAs are involved in regulating the sternness maintenance and differentiation fate decision of neural stem cells,in addition to the important role of histone modification in the regulation of the fate of neural stem cells.Some scholars detected a series of differentially expressed microRNAs by high-throughput microRNAs expression spectrum analysis in neural stem cells from mouse embryonic brain of gestational diabetes.We verified several microRNAs that are closely related to neurodevelopment and mental disease.The expression of mmu-miR-101a-3p had statistical significance,while its function in neurodevelopment is not clear.The expression of mmu-miR-101a-3p in the brain of mice significantly increased from embryonic day 18 to postnatal day 8,in coincidence with gliogenetic differentiation of neural stem cells.There are literature detected up-regulation of hsa-miR-101a-3p in the brain of children suffer from autism and attention deficit/hyperactivity disorder(ADHD).We observed the regulation of mmu-miR-101a-3p and its downstream target molecules Fam60a on transcriptional of GFAP in neural stem cells under differentiated culture condition with high glucose.In this study,we analysed the influence of gestational hyperglycemia on embryonic cerebral cortex development through establishing gestational hyperglycemia mice model and inducing differentiation of primary neural stem cells under the condition of high glucose in vitro.We explored the changes of histone acetylation modification and the related regulation on transcription factors in the early neurogenetic stage of neural stem cells differentiation.Besides,we studied the effect of elevated expression of mmu-miR-101a-3p on astrogliogenesis in the embryonic brain of gestational hyperglycemia mice.Part ? Effects of hyperglycemia in pregnancy on embryonic neurogenesis of neocortexIn order to observe the effect of hyperglycemia in pregnancy on embryonic development of the cerebral cortex in mice,we built hyperglycemic pregnancy mice model to observe the morphology of embryos and do statistical analysis the incidence of embryonic neural tube defects.We found that the incidence of embryonic neural tube defects in moderate hyperglycemia pregnant mice decreased significantly compared with severe hyperglycemia pregnant mice.The moderate hyperglycemia pregnant mice producing more offspring with normal phenotype compared with severe hyperglycemia mice.We observed the neocortex development in these offspring with normalphenotype.At first,we found that the number of CTIP2+ pyramidal neurons in deep layer 5 of the neocortex increased significantly in postnatal 1 day mice affected by gestational hyperglycemia compared with normal pregnancy group;while the number of TBR1+ pyramidal neurons in deep layer 6 and SATB2+ pyramidal neurons in superficial layer 2 to 4 of the neocortex had no significant difference compared with normal pregnancy group.We further observed the embryonic 17.5 day fetuses of gestational hyperglycemia mice.We found that the number of CTIP2+ pyramidal neurons in deep layer 5 of the neocortex increased significantly as well;while the number of TBR1+ pyramidal neurons in deep layer 6 and SATB2+ pyramidal neurons in superficial layer 2 to 4 of the neocortex had no significant difference compared with normal pregnancy group.Otherwise,we found that the thickness of layer 5 incresed in E17.5 embryos and P1 mice affected by maternal hyperglycemia,and more significant in E17.5.The migration of SATB2+neurons seems to be affected by maternal hyperglycemia,related studies may be done in the future.We detected the effects of gestational hyperglycemia on the cell cycle exit of neural stem cells during the early phase of embryonic neocortical neurogenesis through Ki67/EdU dyeing.The results suggests high glucose promotes neural stem cells exit from the cell cycle,and then affects the birth of new neurons,and further affects the distribution of newborn neurons in the deep layer of neocortex.In order to further eliminate effects of gestational hyperglycemia on apoptosis of neural stem cells,we counted the number of apoptosis cells in VZ region and SVZ region of lateral ventricle by TUNEL staining.Results show no significant difference in apoptosis of neural stem cells between embryos of moderate hyperglycemia pregnancy mice and normal pregnancy mice.These results suggest that moderate hyperglycemia during pregnancy affects the production of early-born new neurons by promoting premature exit from the cell cycle in neural stem cells,without significant impact on apoptosis of neural stem cells,which probably further affects the distribution of newborn neurons in the deep layer of neocortex.In order to reveal the effects of high glucose on the advanced cell cycle exit and premature neuronal differentiation of mouse embryonic neural stem cells,we detected the mitosis of neural stem cells under differentiated condition adding high glucose by immunofluorescence staining technique.The results show that high glucose treatment significantly reduced number of neural stem cells Re-entry into the cell cycle(PH3 and EdU mark M phase and S phase of mitosis)under differentiated condition.It suggests increase in the number of neural stem cells exiting from the cell cycle in differentiated culture condition with high glucose level.We further tested the expression of cyclin-dependent kinase inhibitors(CKIs)family factors,P21 and P57.Results show that high glucose level raised the expression of P21 and P57,which might consequently promote the cell cycle exit and the differentiation of embryonic neural stem cells in vitro.We detected the expression of neuronal differentiation marker,MAP2,with qPCR analysis and immunofluorescence staining,and expression of another neuronal differentiation marker,TUJ1,by Western Blotting analysis in the differentiated embryonic neural stem cells treat with high glucose,to verify high glucose treatment promote the differentiation of neural stem cells into neurons.The above experimental results show that maternal hyperglycemia promotes premature cell cycle exit and neuronal differentiation of embryonic neural stem cells,which increases the number of newborn CTIP2+ pyramid neurons and their distribution in deep layer.We further study the mechanism how high glucose promotes premature neuronal differentiation of neural stem cells.Part II Epigenetic Studies on the mechanisms involved in distubed embryonic neurogenesis of neocortex in hyperglycemic pregnancy miceTo reveal the mechanism involved in advanced embryonic neurogenesis of neocortex in hyperglycemic pregnancy mice,we detected the expression of proneuronal and neuronal transcription factors through the qPCR in differentiated neural stem cells in vitro treated with high glucose,as well as in the embryonic cerebral cortex of neurogenesis stage in gestational hyperglycemia mice.Results show that the mRNA level of Ngnl,NeuroD2 increased significantly in the high glucose treated group,compared with control.The mRNA level of Ngn1,NeuroD2 also increased significantly in the embryonic cerebral cortex of neurogenesis stage in gestational hyperglycemia mice compared with normal pregnancy group.In the neurogenesis phase of the neocortex,neural stem cells differentiate towards neurons.In this process,the transcription of proneuronal and neuronal factors is effected by the degree of chromatin density around the promoter region.we observed the nuclear chromatin structure in neural stem cell within the embryonic neocortex of gestational hyperglycemia mice with transmission electron microscopy technology.We found that the number of nuclear heterochromatin increase in neural stem cells within the embryonic neocortex of gestational hyperglycemia mice compared with normal pregnancy group,which suggests chromatin remodeling,histone modification and other complex changes have taken place.Histone acetylation modification is critical for activating transcription of proneuronal and neuronal factors,which is apparent in the neuronal differentiation phase of neural stem cells.Histone acetylation around the promoter region modify transcription of factors associated with neuronal differentiation by influencing opening degree of chromatin nearby and thus affecting the transcriptional activation.We tested the acetylation level of histone H3K9,H3K14,H4K8 and H4K16 in neural stem cell within the embryonic neocortex of gestational hyperglycemia mice with Western Blot technique.The results show that the acetylation level of histone H3K14 increased significantly in neural stem cells of gestational hyperglycemia group,compared with normal pregnancy group,while without obvious differences of acetylation level in other histone sites.At the same time,we tested the acetylation level of above histone sites in embryonic neural stem cells under condition of differentiation treated with high glucose.The results show that the acetylation levels of histone H3K14 increased significantly in high glucose treated group,compared with the control group,while no obvious differences of histone acetylation level of other sites.We further detected enrichment of histone H3K14 acetylation around the promoter region of Ngn1 and NeuroD2 with chromatin immunoprecipitation(ChIP).We found that enrichment of H3K14 acetylation in the promoter region of Ngnl and NeuroD2 increased significantly in gestational hyperglycemia group,compared with normal pregnancy group.The enrichment level of H3K14 acetylation in the promoter region of Ngnl and NeuroD2 also increased significantly in differentiated neural stem cells than the control.As well known,P300/CBP and GCN5 are prime histone acetyltransferases of H3K14ac,SIRT1 is histone deacetylase.They participate in regulating fate decision of neural stem cell.We tested the expression of CBP/P300,GCN5,SIRT1 in mRNA level and protein levels in differentiated neural stem cells after exposure to high glucose,and in neural stem cells from gestational hyperglycemia mice under differentiation culture conditions.Results show that the expression of SIRT1 in the mRNA level and protein level was lower in above two experimental groups than the control group.The expression of P300 in protein level was higher than the control group.The expression of CBP and GCN5 in mRNA level and protein level had no significant statistical difference.Results suggest high glucose may inhibit histone deacetylase activity of SIRT1 and increase the histone acetyltransferases activity of P300,thus increasing histone H3K14 acetylation.Together,these results suggest that maternal hyperglycemia promote the neural differentiation of neural stem cells by promoting the cell cycle exit,and by activating transcription of Ngnl and NeuroD2 via increasing the enrichment of histone H3K14ac in the promoter region of Ngnl and NeuroD2.Which probably affect neocortical neurogenesis and the distribution of ealy-born neurons in the deep layer 5 of neocortex.Part ? Role and mechanism of mmu-miR-101a-3p regulated the premature astrogliogenesis of neural stem cells in hyperglycemic conditionWe found that maternal hyperglycemia disturb neocortical astrogliogenesis.Astrocytes play an important role in synaptic formation and signal transmission.Some microRNAs are involved in regulating the corticogenesis and fate determination of NSCs.Maternal hyperglycemia disturbs the astrogliogenesis by unknown mechanism.We detected expression of microRNAs differentially expressed in the brain of embryos of gestational hyperglycemia mice,referencing the results of high-throughput microRNAs expression spectrum analysis,which also participate in the regulation of fate decision of neural stem cell differentiation,or closely related to neurodevelopment and Neuropsychiatric disorders.We found the expression of mmu-miR-101a-3p increased significantly in the embryonic brain of gestational hyperglycemia mice.Literature shows that the expression of mmu-miR-101a-3p increased gradually from the late embryonic stage to the early postnatal days in the brain.The expression of hsa-miR-101a-3p is upregulated in the autistic and ADHD.To study the effect of mmu-miR-101a-3p and high glucose exposure on astrogliogenesis in vitro,we firstly confirmed the higher expression of mmu-miR-101a-3p and premature astrocyte in high glucose treated differentiated neural stem cells.We transfected neural stem cells with mmu-miR-101a-3p mimic,the elevated expression of mmu-miR-101a-3p promoted the neural stem cells differentiated into astrocyte under the differentiation culture condition.The results suggest that elevated expression of mmu-miR-101a-3p,consistent with high glucose exposure,promotes astrogliogenesis.We detected expression of predicted target genes of mmu-miR-101a-3p in embryonic brain from gestational hyperglycemia mice.We found the expression of Sinhcaf,which encodes the protein Fam60a,decreased in embryonic brain from gestational hyperglycemia mice.After the expression of mmu-miR-101a-3p was inhibited,the expression of its target gene Sinhcaf became elevated.We need further detection on combination of mmu-miR-101a-3p with 3 'UTR of Sinhcaf by dual luciferase assay.In order to reverse validate mmu-miR-101a-3p and high glucose exposure affect astrogliogenesis through inhibit expression of Fam60a protein.We plan to transfect neural stem cells with Fam60a over-expression plasmid,and then cultured in differentiation conditions.Fam60a is known as a key component of Sin3a-HDAC complexes,which is critical for complex aggregation.And an article about the combination of Sin3a-HDAC complexes and GFAP promoter region inhibits histone acetylation in the promoter region of GFAP and transcriptional activation of GFAP.We detected combination of H3K14ac on promoter regions of GFAP through ChIP technology in embryonic dorsal telencephalon tissue of pregnant mice with hyperglycemia.The results show that maternal hyperglycemia increased the combination of H3K14ac on promoter regions of GFAP.Based on these results,we predict that expression of mmu-miR-101 a-3p is upregulated in embryonic brain from gestational hyperglycemia mice,it combines with 3 'UTR of Sinhcaf,thereby reducing expression of protein Fam60a,which accordingly influence the combination of Sin3a-HDAC complexes on promoter region of GFAP,therefore increasing combination of H3K14ac on promoter region and promoting transcriptional activation of GFAP,ultimately these modifications promote the premature differentiation of neural stem cells into astrocytes.CONCLUSIONModerate hyperglycemia in pregnancy promotes premature exit from the cell cycle in neural stem cells,and increases the histone H3K14ac and its enrichment in the promoter region of Ngnl and NeuroD2,mediating the transcriptional activation of Ngn1 and NeuroD2,thus promoting premature neurogenesis,ultimately probably affect the distribution of earlu-born CTIP2+ neurons in the layer 5 of neocortex in embryos of gestational hyperglycemia mice.The expression of mmu-miR-101a-3p increased significantly in embryonic brain of gestational hyperglycemia mice,which increase astrocytes differentiation.The role and mechanism of Fam60a in astrocyte need further detection.It is beneficial to find the key mechanisms regulating abnormal neural stem cell differentiation caused by maternal hyperglycemia,providing possible guidance for screening neurodevelopment disorders caused by gestational hyperglycemia.Considering that epigenetic modifications is reversible,it can be realized to reverse changes in epigenetic modifications through regulating related enzyme.Therefore,seeking out key epigenetic factors,also provide possible intervention and reversal for embryonic neurodevelopment disorders caused by gestational hyperglycemia.The methods in this study can also be used to study effectes of other maternal adverse factors or environmental factors on the neurodevelopment of offspring.Innovation:1.This project first discovered that hyperglycemia during pregnancy affect embryonic neocortical neurogenesis,then the number and distribution of new-born neurons.2.We first found that regulation of histone acetylation on proneual and neuronal factor participate in the regulation of premature neurogenetic differentiation of neural stem cells affected by maternal hyperglycemia.3.We first discovered that the regulation of mmu-miR-101a-3p is involved in regulating premature astrocytes differentiation in embryonic brain affected by gestational hyperglycemia. |
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