The Mechanism Of Tctn3 Knockout Causing Apoptosis And Neural Tube Defects In Mice

Background and Objective:Ciliary diseases are a kind of systemic diseases caused by ciliary dysfunction.To date there are nearly 200 genes have been found to be associated with ciliary diseases.Among these genes,Tectonic(TCTN)gene mutation is an important cause of some primary ciliary diseases.TCTN gene family includes three members: TCTN1,TCTN2 and TCTN3.The TCTN proteins encoded by these genes are mainly distributed in the transition region of cilia and are involved in the formation of cilia and the regulation of some signaling pathways such as sonic hedgelog(Shh).Mutations in the TCTN gene family can lead to many ciliary diseases,such as Joubert syndrome(JBTS),Bardet-Biedl syndrome(BBS),Oral-facial-digital syndrome(OFDS),Meckel-Gruber syndrome(MKS)and so on.Previous studies have shown that TCTN1 and TCTN2 gene mutations can lead to neural tube defects and brain abnormalities in human and mice,but the role of TCTN3 gene in brain development is still unclear.Therefore,in this study,we used the Tctn3 knockout mice produced by piggy Bac transposon system to explore the effects of Tctn3 on neural tube development and Shh signaling pathway,and to explore the mechanism of regulating cell death.Methods:Tctn3 knockout mice produced by piggy Bac transposon system were cultivated,Quantitative PCR(Q-PCR)was used to detect the m RNA expression of Tctn1,Tctn2,and Tctn3 in Tctn3 knockout(KO)mice and Tctn3 heterozygous KO(Het)mice brain tissues of 10.5(E10.5)days;to observe the appearance and bone staining of the surviving mice after birth,and to observe the effect of Tctn3 KO on the development of mice;the expression and distribution of neural tube development related proteins in E10.5 Tctn3 KO mice and Tctn3 Het mice were observed by immunofluorescence staining;the brain tissues of E10.5 mice were collected for RNA sequencing analysis,and the m RNA expression of Shh signaling pathway related genes in the brain tissues of Tctn3 KO mice on E12.5 days was verified by Q-PCR;immunofluorescence staining was used to detect the number of neurons in E12.5 Tctn3 KO mice,western blot was used to detect the changes of Bcl2,Bax,cleaved PARP1,PI3K/Akt signaling pathway and m TOR signaling pathway regulated by Shh signaling pathway;Primary embryonic fibroblasts(p MEFs)were cultured,the apoptosis of Tctn3 KO mice p MEFs was detected by flow cytometry,and the effect of Akt specific agonist SC79 on apoptosis was detected;to explore the regulatory mechanism of Tctn3,the expression of Nphp1(anti-apoptotic protein in ciliary transition region)in E12.5 mouse embryonic brain was detected by Western blot,and the interaction between Tctn3 and Nphp1 was detected by Co-immunoprecipitation(Co-IP);The NIH3T3 cells were transfected with Nphp1 plasmid,to observe the effect of Nphp1 overexpression on apoptosis,Shh signal and PI3K/Akt signal of Tctn3 knockdown cells.Results:Our results showed that the expression of Tctn3 m RNA was not detected in the Tctn3 KO mice,which confirmed that the gene was knocked out in the mice,and Tctn3 gene knockout did not affect the m RNA expression of Tctn1 and Tctn2;statistics showed that almost half of the Tctn3 KO mice died of E14.5,Tctn3 KO mice showed abnormal development of the whole body,including eyeball diminution,eyelid closure,polydactyly,and embryonic death;bone staining showed that the head of Tctn3 KO mice was dome shaped and incomplete closure,decreased anteroposterior length length and abnormal sternal fusion.These results suggest that the knockout of tctn3 gene causes developmental malformation and embryo death in mice.Immunofluorescence staining showed that the expression of Shh and Foxa2 in the ventral neural tube of E10.5 Tctn3 KO mouse embryo was significantly decreased,and the expression regions of neural tube markers Nkx2.2,Olig2,Hb9,Isl1/2 and Pax6 were changed and moved to the ventral side,while Pax3 expression remained unchanged.In particular,the expression of the cilia specific marker protein Arl13 b was significantly decreased.After RNA sequencing,KEGG enrichment analysis and cluster analysis showed that Shh and Shh signaling pathway related genes,such as Gli1 and ptch2,were significantly down regulated in Tctn3 KO mice.Q-PCR results further confirmed that Gli1 and Ptch1 m RNA expression levels downstream of Shh signaling pathway were significantly decreased in Tctn3 KO mice.These results suggested that Tctn3 knockout affect the expression of Shh signaling pathway related genes.We also found that the number of Neu N positive cells in the brain tissue of Tctn3 KO mice was significantly decreased,and the expression of anti-apoptotic protein Bcl2 was significantly decreased,while the cleaved PARP-1 and pro-apoptotic protein Bax were significantly increased.Flow cytometry showed that there was significant apoptosis in the cultured primary embryonic fibroblasts of Tctn3 KO mice;SC79,a specific activator of Akt,significantly reduced the number of apoptotic cells,suggesting that Tctn3 gene knockout induced PI3K/Akt dependent apoptosis.Because of the existence of nphp1 in the ciliary transition region and its anti-apoptotic effect,we found that the content of Nphp1 in E12.5 Tctn3 KO mouse embryonic brain tissue was significantly decreased.Immunoprecipitation results showed that there might be interaction between Tctn3 and Nphp1.Overexpression of Nphp1 inhibited the apoptosis of Tctn3 knockdown cells and reversed the decrease of Shh signal and PI3K/Akt signal,which confirmed that Tctn3 may play an anti-apoptotic role by forming a complex with Nphp1.Conclusions:The knockout of Tctn3 gene inhibited the activation of Shh signaling pathway and caused embryonic dysplasia and embryonic death in mice,especially affected neural tube development and brain dysplasia.Tctn3 gene knockout induced the increase of neuronal apoptosis;as a protein co-expressed in the ciliary transition region with Tctn3,Nphp1 can form a protein complex with Tctn3 and participate in the regulation of neuronal apoptosis.To explore the function of Tctn3 protein and its pathogenesis in ciliary diseases will contribute to the diagnosis and treatment of ciliary diseases and improve the quality of life of patients.