The Effect Of Mutant P53^N236S In The Development Of Mouse Embryonic Nervous System

The development of embryonic nervous system is a very complicated and orderly process. In most vertebrates, oosperm can form the three germ layers through cleavage. Ectoderm develops into the central nervous system. First, ectodermal cells in the back midline region proliferate and form neural plate. Next, neural plate begins to merge into the midline position by the influence of the notochord and adjacent mesoderm signals. After neural groove and the neural fold period, neural plate eventually leaves the surface ectoderm and formed closure hollow neural tube. Neural tube closure occurs along A-P axis regularly. Anterior neuropore closes and enlarges into brain. Posterior neuropore closes, and develops into the spinal cord. Neural tube closure disorders leads to neural tube defects in embryonic development. Anterior neuropore dysraphism results in malformations. Posterior neuropore dysraphism causes spinal bifida. Moreover, the neural crest cells near to dorsal neural tube migrate to both sides of the neural tube and forms dorsal root ganglion. Neural crest cells on both sides of the neural tube develope into the peripheral nervous system. Molecular pathways and regulatory mechanisms of neural tube in various development stage are very compleicated.The p53 gene is an important tumor suppressor gene. Increasing data suggests that p53 plays an important role in embryonic development, In mouse embryo before 10.5dpc, p53 is ubiquitously expressed. In 12.5dpc, p53 and MDM2, the key protein regulating p53 degradation, coexpressed in neural tube. The p53 presents low expression in the late terminal differentiated organization. These data suggest that p53 can regulate embryonic development. Studies have shown thatp53-/-female embryos displayed severe brain malformations.p53N236S (p53N239S in human, refer as p53S) was found in premature senescence cells (G5mTR-/-Wrn-/-) that escaped senescence and converted to tumor. It is a missense point mutation occurred in p53 DNA binding domain, and was one of p53 hot spot mutations.Our laboratory had established p53s/s mouse model in previous work and had set p53s/s♂xp53s/+♀ to produce offsprings. By analyzing offspring genotypes and gender distribution, we found that the number of mice with p53s/s genotype is significantly less than mice with p53s/+genotype, especially female mice. These statistics results suggest that p53s/s female embryoes was dead during embryonic development. The p53N236S homozygous mutation might affect the embryonic development of p53s/s female mouse, which led to its low birth rate.Based on the above hypothesis, in this study, we used p53s/s♂Xp53s/+♀ and p53s/+S♂ x p53s/+♀ mating strategy to get 8.5dpc to 13.5dpc embryos and analyzed embryonic genotype and gender. We found that the number of p53s/s and p53s/+fetuses had no significant difference before 13.5dpc. The results show that embryonic lethality may have occurred after 13.5dpc. We found that p53s/s genotype female embryos have a large number of neural tube defects.There are about 73%of the embryos manifested anencephaly and 13%of the embryos had spina bifida, but it rarely appeared in the male p53s/s embryos and p53s/+embryos.The probability of anencephaly is higher in p53s/s female embryos than p53-/- female embryos. These data suggested that p53S mutation gained the fuction in leading to anencephaly. By analyzing the paraffin sections of 10.5-13.5dpc anencephaly and spina bifida embryos, we found that the neural tubes did not close in the location of anencephaly and spina bifida. Since wild-type p53 inhibited cellular proliferation, and p53S mutation led to abnormal cell proliferation, we speculated that the abnormal proliferation of nerve cells may cause neural tube defects.Because of the important role of DNA helicase Wrn gene in DNA replication, recombination, repair and telomere stability, Wrn knockout can lead to cell proliferation inhibition. We use Wrn knockout mice to cross with p53S mice to study whether Wrn-/-can reverse neural tube closure defect caused by p53S mutation. Our found the number of female mice with Wrn-/-p53s/s genotype is less than male. The Wrn-/-p53s/s female embryos showed brain malformation and spina bifida, but the anencephaly probability (38%) was significantly lower than p53s/s female embryos (73%), suggesting that Wrn-/-partially rescued the anencephaly caused by p53s/s.We further studied the proliferation of neural tube cells from 10.5dpc and 11.5dpc by BrdU incorporation assay. The results showed that the proliferation rate of misclosure neural tube was significantly higher than the adjacent normal closure neural tube and the wild-type neural tube at similar position. The results supported that the abnormal proliferation of cells in the neural tube led to female embryonic neural tube defects in p53s/s mice, and thus led to p53s/s female embryonic lethality.In addition, by HE staining of paraffin sections, we found that neural tube from female p53s/s embryos displayed multiple dorsal root ganglia phenotype. This phenomenon is associated with spina bifida and anencephaly phenotype. The p53s/s may affect the migration or proliferation of neural crest cells and dorsal root ganglia formation. This study reported the preliminary mechanism for p53S mutation induced embryonic lethality, provided an important basis for studying p53 function in nervous system development.