Research On The Function And Mechanism Of PDAP1 In Mouse Embryonic Development

Background and purpose:Large-scale phenotypic studies have shown that approximately 25%-30%of mouse gene knockouts result in embryonic death.Phenotypic studies of these knockout lines focused more on the embryo itself,ignoring organs outside the embryo,such as the yolk sac and placenta.From the second trimester,the placenta is an important nutrient supply organ.Placental malformations were present in 68%of knockout lines that were fatal in or after the second trimester,and second-trimester deaths（E9.5–E14.5）was almost always associated with severe placental malformations.The yolk sac is an important extraembryonic structure that provides nutrition for embryos in the first trimester（E7.5-E9.5）,and the morphological analysis of the yolk sac of 66 genetically deficient mice found that 11%of the yolk sac morphology was abnormal,and the embryos with yolk sac abnormalities died in the E9.5-E14.5 stage.Platelet-derived growth factor associated protein 1（PDAP1）is a phosphorylated protein with a molecular weight of 28 k Da,stable to heat and acid,which was first isolated and purified in rat brain tissue.PDAP1 is associated with tumor cell proliferation and is highly expressed in various malignancies such as childhood acute lymphoblastic leukemia,glioma,gastric cancer,and rectal cancer.In the previous study,we found that PDAP1 promotes the occurrence and development of colon cancer.In the process of constructing a Pdap1 knockout mouse model,it was found that Pdap1knockout was embryonic lethal,indicating that Pdap1 is involved in regulating mouse embryo development,however,the underlying mechanisms remain unsolved.Therefore,on this basis,this study determined the time of embryo death and dysmorphologies of the Pdap1^-/-embryo and revealed the underlying mechanisms,which provided new evidence for elucidating the regulatory mechanism of mammalian embryonic development.Materials and methods:Spatial and temporal expression of PDAP1 protein in mouse embryos was determined by immunohistochemistry.Pdap1 knockout mouse was generated by CRISPR-Cas9 and genotyped by PCR and sequencing.Immunohistochemistry and western blotting were performed to validate PDAP1expression.By analyzing the survival status of Pdap1^-/-embryos in different developmental phases,the time of embryo death was clarified.Stereo microscopy and HE staining were used to observe the Pdap1^-/-embryonic morphological abnormalities.The molecular mechanism of abnormal embryonic development caused by Pdap1deletion was determined by immunohistochemical staining,immunofluorescence staining,proteomics,transcriptomics analysis,and cellular-level experiments.Results:1.The results of immunohistochemical staining of wild-type embryos showed that the expression of PDAP1 protein in E10.5-E14.5 stage embryos showed an increasing trend.Pdap1^+/-heterozygous female and male mice were developed without abnormalities and were fertile.2.The results of genotype analysis of 323 offspring produced by 46 groups of Pdap1^+/-heterozygous mice showed that Pdap1 homozygous embryos were lethal.The results of genotype identification and morphological analysis of 173 embryos obtained from E9.5-E13.5 showed that Pdap1^-/-embryos died from E10.5-E11.5.3.The morphological and histomorphological analysis results of Pdap1^-/-embryos showed that Pdap1^-/-embryonic development was severely delayed,and there was no abnormal development of neural tubes,brains,body segments,and other organs,pericardial enlargement,and pericardial space hemorrhage.The results of pathological analysis of the placenta and yolk sac showed that the Pdap1^-/-placental labyrinth decreased or even disappeared,the Pdap1^-/-placental labyrinth vascular was abnormal,and the Pdap1^-/-yolk sac was abnormal vascularized.4.The results of proteomics,transcriptomics,and cellular-level experiments showed that PDAP1 further promoted vascularization by downregulating NID-1expression to maintain vascular endothelial cell migration,invasion,and angiogenesis.Conclusion:PDAP1 promotes yolk sac angiogenesis by downregulating NID-1expression;Abnormal yolk sac vascularization of Pdap1^-/-embryos in stages E10.5-E11.5 may be one of the causes of embryonic death.