Preliminary Study On The Mechanism Of P53^N236S Leading To Neural Tube Defects

Neural tube defects(NTDs)are a serious birth defect,caused by the failure of neural tube closure.NTDs occur in 1/1000 human births in the world.Mouse neural tube closure occurrs during 8.5～10.5dpc.In the beginning,the neural epithelium differentiation into neural progenitor cells to form the neural plate,subsequently followed by contraction of the neuroepithelium to make the neural plate bend in the the dorsal midline to form neural folds.Then the neural folds bend at the dorsolateral hinge point,which fuses at the midline to form the neural tube.NTDs are divided into exencephaly,craniorachischisis and spina bifda.P53N236S(hereinafter referred to as p53S,homozygous mutation to p53s/s)is found in the premature cells(G5mTR-/-Wrn-/-escape aging and tumorigenesis process,apoint mutation occurred in the p53 DNA binding region,the 236 codon of the p53 protein mutated from asparagine to serine.Previous studies have shown that p53s/s mouse embryos displayed NTDs phenotype,such as the exencephaly and spina bifida.This article mainly studied the new gain of function of p53 mutation(p53N236s)in the process of neural tube closure.In order to further validate this phenotype,We dissected 9.5dpc,10.5dpc,12.5dpc,13.5dpc and 16.5dpc mouse embryos,usingp53s/s ♂×p53s/+♀mating,respectively,and found 116 female embryos with NTDs in 156p53s/s totle female embryos,accounting for 74.3%of p53s/s female embryos.Under the same feeding condition,the/p53-/-NTDs embryos accounted for 38.88%.(14/36)of p53-/-female embryos,23%reported in the literature,and the incidence of NTDs in p53s/s femaic embryos was much higher than that of p53-/-,suggesting that p53S has new gain of function in the process of neural tube closure.In 9.5dpc-13.5dpc,p53s/s NTDs were paraffin embedding,sliced and stained with HE.It was found that the female NTDs embryos showed openingneural tube,and with the increase in the number of days the degree of closure is gradually increased.At the same time,exencephaly of the female embryos ventricular atrophy,which may be due to excessive proliferation of neural cell.Therefore,we used BrdU incorporation and pH3 immunohistochemistry to detect the proliferation of neuronsin p535s/sNTDs and WTembryos in 9.5dpc and 10.5dpc.In 9.5dpc,in the BrdU incorporation rate was 50%,pH3 positive rate of 9%inp53s/s NTDs,significantly higher than wild-type embryos 30%and 5%.In 10.5dpc,the positive rate of BrdU incorporation was 45%,the positive rate of pH3 was 8%in p53s/sNTDs,also higher than wild-type embryos 30%and 4%.These dataindicated that p53S leads to abnormal proliferation neural cells.Combined with previous experimental data,the incidence of NTDs in Wrn-/-p53s/s female embryos was 38%,suggesting that knockout of Wrn gene in p53S background inhibitedcell proliferation,can rescue the occurrence of NTDs.Detection of Wrn-/-p53s/s NTDs neuronal proliferation levels in 10.5dpc,we found the proliferation rate of Wrn-/-p53s/s NTDs(the positive rate of BrdU incorporation was 35%and the positive rate of pH3 was 6%)was lower than that in p53s/s NTDs.Further showed that p53S inducedneural cell abnormal proliferation,inhibited of neural tube closure.At the same time,we also examined the proliferation of neural cellin p53-/-NTDs,found the proliferation of neural cell(the positive rate of BrdU incorporation was 46%and the positive rate of pH3 was 7%)in p53-/-NTDs female embryoswas significantly higher than that in WT(the positive rate of BrdU incorporation was 30%and the positive rate of pH3 was 4%).But there was no significait difference in proliferation rate compared with p53s/s NTDs(BrdU incorporation positive rate of 45%,pH3 positive rate of 8%).These data suggested that p53S leaded to abnormal proliferation of neural cells,affected the the neural tubeclosurethrough p53 loss of function.In the process of neural tube closure,the proliferation and differentiation of neuralprogenitor cells is a relatively balance process.The proliferation of p53s/sNTDs neurons is abnormally elevated and may affect the differentiation of neural cells.Furthermore,we used Tuj1 immunohistochemistry to detect the differentiation of p533s/sNTDs.Tujl was expressed in 9.5dpc WT and p53-/-NTDs embryos,whereas Tuj1 was not detectedin p53s/s NTDs embryos.The positive rate of Tuj1(5%)in 10.5dpc p53s/s NTDs embryo relative to WT(12%)and p53-/-NTDs(12%)were significantly reduced,suggesting that p53S delayedneural cells differentiation through p53 gain of function.In the experiment,we found that the birth rate of p53s/s female was about 25.5%,and there was no abnormal phenotype.We used the behavioral experiments of elevated plus maze,suspension and so on to detect the anxiety and depression ofp53s/sfemale mice and males.In the open field experiment,it was found that p53s/s female was more anxious than WT female,but there was no significant difference in anxiety level of p53s/s male mice.Suspension test showed that compared with the fixed time of WT female(204.1±21.62s),p53s/s females were significantly elevated(295.4±11.76s),indicating that p53s/s females were more depressive than WT female mice.P53 not only triggers the NTDs phenotype in female embryos,but also affects the development of female embryonic spine.Alcian Blue Staining was used to detect the bones of p53s/s embryos in 13.5dpc,found that p53s/s female embryos displayed vertebral fusion phenomenon,the incidence of female embryos accounted for about 35%(7/20).PET/CT detection of p53s/s adult skeleton,also found one vertebral fusion phenotype(1/5).p53S affects the spine development of mice and has sex basis.At the same time we also detected the skeletal of p53-/-embryos and found that p53-/-female embryos also had vertebral fusion,the incidence was about 33.3%(1/3).qRT-PCR showed that the expression levels of BMP2,BMP3,BMP4,Smad2,Runx2 and Osx in p53s/NTDsand p55/-NTDsembryos were significantly decreased,compared with WT embryos,suggesting that p53S mayinhibit the development of bone by inhibiting the BMP pathwaythrough p53 loss of function.In summary,p53S homozygous mutation not only triggers the NTDs phenotype in female embryos,but also affects the development of its spine.On the one hand,p53S may promot the proliferation of neuronal cells and delay neuronal differentiation,leading to the occurrence of NTDs.On the other hand,p53S may affect the development of the spine by inhibiting the BMP pathway.Behavioral experiments found that p53 s/s female mice showed more anxiety and depression.