Ciliogenesis And Hedgehog Signaling Pathway

The primary cilium is a non-motile microtube-based protrusion from the cell surface and is found on most vertebrate cells.The primary cilium serves not only as a key mechanosensory organelle but also as a site for signal transduction for several signaling pathways,including the Hedgehog?Hh?pathway.And the defects in cilia structure and function result in a wide spectrum of structural birth abnormalities,which are collectively known as ciliopathies.Interestingly,many of these defects are due to the disruption of Hh signaling.The Hh family of secreted signaling proteins plays basic roles in cell fate specification,proliferation,and differentiation.Loss of Hh signaling results in a wide range of birth defects,whereas aberrant activation of the Hh pathway causes several types of human cancer.Therefore,understanding the molecular mechanism of ciliogenesis and relationship between cilia and Hh signal are urgent problems to be solved,which are crucial for the study and treatment of related disease.Based on the report and the study results in our lab,we chose three cilia related protein as research objects:Centrosome protein Talpid3?Ta3?,previous study showed that Ta3 mutation resulted in reduced ciliogenesis and Hh activity,but the mechanism of this is not clear.In addition,activation of transcriptional factor Gli protein by Hh occurs within cilia,however there is still no evidence proved this hypothesis;Cilia transition zone protein Tectonic3?Tctn3?is the homolog of Tctn1 and Tctn2,which found in human gene only.All of them affect the ciliogenesis and Hh signal,but the relationship between them is not clear;Basal body appendages protein Dzip1l is a mammalian homolog of zebra fish Iguana?Igu?,which reportedly affect cilia function and Hh signal,but does not affect ciliogenesis.So we would like to know the exact role of Dzip1l in ciliogenesis.Based on these questions,the mainly study work of this dissertation is performed as the following aspects:1.Firstly we constructed the Ta3 mutant mice and got the homozygotes embryo and cells.Then observation the ciliogenesis and Hh depended neural tube pattering effected by Ta3 with immunofluorescence staining.And then the level of Gli2,Gli3 and pGli in embryos and cells was checked by Western Blot to determine the effect of Ta3 or drugs on Hh signaling pathway activity.Lastly,we constructed several Ta3 mutants in different length,and then analyzed the relationship between Ta3 and PKAR,and the localization of Ta3 to centrosome by immuno-precipitation and cell staining.Through a series of experiments and analysis,we directly demonstrated that the dephosphorylation and activation of Gli2 and Gli3 are performed within the cilia;The decrease of Gli2 and Gli3 process in Ta3 mutant was due to the decreased phosphorylation level of Gli2 and Gli3;N-terminal of Ta3 is necessary for its localization to the centrosome and C-terminal is essential for its binding to PKAR;it is the first time we found that PKAR miss localized in many Ta3 mutant cells,which may be one reason for the reduced Gli3 phosphorylation level.2.We checked the phenotype of Ta3 homozygous embryo by construction the mutant mice.And then the effect of Tctn3 mutant on ciliogenesis and Hh signal by embryo section and cell staining was checked.Subsequently,the Tctn1 and Tctn2 gene were knocked in Tctn3 site respectively and got corresponding mutant embryos.Based on the embryos and cells,the ciliogenesis,neural tube pattering and Gli protein level were detected.At the same time we detected the ciliogenesis in Tctn3 mutant cells by expression exogenous Tctn1,Tctn2 and Tctn3.In addition,we checked the relationship between three Tctn proteins and other known transition zone proteins by immune precipitation and cell staining.To date,we report that:Loss of Tctn3 gene function in mice results in a decrease in ciliogenesis and Hh signaling;The phenotypes of Tctn3mutant are similar to those in knock in mouse;Overexpression of Tctn3,but not Tctn1 or Tctn2,can rescue ciliogenesis in Tctn3 mutant cells;Similarly,replacement of Tctn3 with Tctn1 or Tctn2in the Tctn3 gene locus results in reduced ciliogenesis,holoprosencephaly,and randomized heart looping,however,the neural tube patterning and the Gli3^Rep process,which both are usually impaired in ciliary gene mutants,are normal;This is the first example of a ciliary gene manipulation that separates ciliogenesis from Hh signaling.3.We generated the Dzip1l mutant allele by knockout technology,and got the phenotype of Dzip1l mutant mouse.We can see the neural tube pattering by immunostaing the embryo section,which may reflect its affection to Hh signal pathway.And then we observed the super-structure of cilia by Scanning and transmission electron microscopy.It is sure that Dzip1l is required for cilia genesis.Next,Dzip1l's localization in cilia was confirmed by cell staining.Several Dzip1l-related proteins were obtained through immuno-affinity purification and mass spectrometry analysis.And their structure relationship was confirmed by immunoprecipitation.Based on this,single mutant and double mutant embryos of Dzip1l-^/+;Bromi-^/+and Dzip1l-^/+;Cby-^/+were obtained through mouse cross reactions.Lastly we detected the embryos'phenotype,the neural tube pattering,cilia's number and morphology in the neural tube and mesenchymal cells,the characteristics of ciliogenesis at different stages by TEM.Here,we show that:Loss of Dzip1l results in significantly reduced ciliogenesis and Hh signal in vivo;Dzip1l interacts with and acts upstream of Cby,an appendage protein,in ciliogenesis;Dzip1l is required for recruitment of Rpgrip1l to the transition zone and removal of Cp110 from mother centriole.Finally,we made a conclusion that Dzip1l promotes ciliary bud formation by controlling the integrity of transition zone and removal of Cp110 from mother centriole in ciliogenesis.In summary,we studied the three cilia related proteins Ta3,Tctn3,and Dzip1l through mouse,embryo,cell,and molecular levels and found their role in ciliogenesis and their effect on Hh signaling.It is hoped that our work of this paper can provide references for the study of subsequent ciliogenesis mechanism and provide theoretical basis for the study of related diseases and screening of drug targets.