Exploring The Role Of Zbed3 In Mouse Cortical Development Using Knockout Mice

With the progress of evolution,the structure of the brain has undergone tremendous changes.From the reptilian cortex to the mammalian neocortex,the function of the brain has achieved a huge leap from maintaining simple body biological functions to having dramatic emotional changes.The neocortex of the mammalian brain is composed of 6 layers of neurons and glial cells.The molecular characteristics,cell morphology,and fiber projection position of each layer of neurons are greatly different.In the past few decades,technological advances and improvements of research methods have promoted our understanding of the development of the nervous system,but the regulation mechanisms of mammalian neocortex development remain unclear.In terms of current understanding,signal molecules,transcription factors,and epigenetic regulation play an important role in the development of mammalian cerebral cortex.In mammals,the canonical Wnt(Wnt/?-catenin)signaling pathway is highly evolutionarily conserved.The canonical Wnt signaling pathway is involved in multiple processes of embryonic development,and plays an important role in the early development of the nervous system and the formation of neocortex.Zbed3 is a member of the zinc finger Bed domain superfamily,can bind to Axinl through "PPPPSPT" motif,and then regulates the level of ?-catenin,which is a modulator participating in the regulation of the canonical Wnt signaling pathway.The work from Dr.Ruan Xiangbin of our laboratory demonstrated that Zbed3 could participate in the regulation of canonical Wnt signals through in utero electroporation,and in the regulation of the formation of cerebral cortex during the development of mouse cortex.In order to further study the role of Zbed3 in the developmental neocortex in mice,we introduced Zbed3 conventional knockout mice from the professor Li Lei of the Institute of Zoology,Chinese Academy of Sciences.The development of Zbed3 conventional knockout mice were normal,and no shortened life span and abnormal behavior were observed.Therefore,we further analyzed the effect of Zbed3 knockout on the nervous system.Comparing the whole brain MRI results of Zbed3 conventional knockout mice and control mice,it was found that there were no obvious defects of brain structure in Zbed3 conventional knockout mice.Next,we analyzed the effect of Zbed3 knockout on proliferation of neural progenitor and cortical layer formation.Through EdU incorporation experiment(30 min),combined with neural progenitor cell marker Pax6,we found that Zbed3 knockout did not affect the proliferation and stemness maintenance of neural progenitor in E12.5,E14.5 and E16.5 mice.Through EdU birth-dating experiments(E12.5 and E14.5),combining the deep cortical marker Sox5(layers ? and ?)and the superfacial cortical marker Cuxl(layers ?,?,?),we analyze the cortical layer formation and neuronal migration in Zbed3 conventional knockout mice.The results showed no significant difference between Zbed3 conventional knockout mice and control mice.With the application of reverse genetics technology,it has been found that knockout and knockdown of the same gene will produce different phenotypes.In order to determine whether there is genetic compensation after Zbed3 knockout,we used qPCR technology to detect the mRNA expression of Wnt-related genes in the control group and the Zbed3 knockout group.We found expression of Axinl and ?-catenin changes in Zbed3 knockout mice.We further tested Axinl and ?-catenin proteins by Western Blot,and found that ?-catenin protein levels were elevated in Zbed3 knockout mice.In order to investigate the upregulation of ?-catenin,we analyzed phosphorylation of ?-catenin and the subsequent processes involved in phosphorylation of ?-catenin could be degradation or Wnt/?-catenin target genes transcription activation,which depends on the phosphorylation of different residues.We further tested several phosphorylated forms of ?-catenin,and found that both the degraded phosphorylated ?-catenin(Ser33/37/Thr41)and the phosphorylated ?-catenin(Ser552 and Ser675)involved in transcriptional activation were found at the protein level.Rise.In conclusion,we analyzed the effect of Zbed3 knockout on the development of neocortex through Zbed3 conventional knockout mice.Due to the limitations of in utero electroporation technology and the characteristics of the cell autonomous feature of canonical Wnt signaling pathway,we have not observed the apparent phenotype of Zbed3 conventional knockout mice on the developmental neocortex.However,we subsequently found that ?-catenin was elevated in Zbed3 conventional knockout mice,which reflected the functional correlation between Zbed3 and ?-catenin,and also implied the functional importance of Zbed3.