2020 Fiscal Year Research-status Report
Functional investigation of Cables2, a novel transcription cofactor regulating Nanog expression through Smad2 activation, during germ cell development
| Project/Area Number |
19K16020
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| Research Institution | University of Tsukuba |
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Principal Investigator |
DINH T.H.TRA 筑波大学, 医学医療系, 助教 (30816476)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | Cables2 / germ cell / embryogenesis |
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| Outline of Annual Research Achievements |
Primordial germ cell (PGC) development is one of the most important biological process in which Nanog and Smad2 contribute into the germ cell specification during gastrulation. Cables2 is expressed in mouse embryonic and adult tissues with the highest RNA level in testis, suggesting the existence and contribution of Cables2 in male germ cell lineage. Furthermore, Cables2 was recently found to involve in Nodal/Smad2 signaling pathways in regulating Nanog expression in mouse gastrulation. Therefore, the temporal function of Cables2 and the Cables2-binding protein facilitating Cables2-Smad2/Nanog in PGC and germ cells development should be discovered.
The conventional Cables2 knock-out (KO) embryos are arrested the normal development from E6.5. Therefore, the Cre/loxP system will be applied to analyze the PGC and germ cell-specific function of Cables2. Using the CRISPR/Cas9 system, the floxed Cables2 mouse with loxP sites flanking exon1 was successfully generated. The floxed Cables2 mice were mated with Nanos3-Cre and Gdf9-Cre mouse lines to examine the function of Cables2 in PGC and germ cell development.
The reporter mice of Cables2 showed the widespread expression of Cables2 in mouse tissues and with high intensity in brain, testis and ovary. Furthermore, immunoprecipitation analysis using the brain and testis of Cables2 reporter mice revealed interaction of Cables2 with CDK5. Collectively, the new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function in both male and female reproductive organs.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Due to lacking of commercially valuable Cables2 antibody, the bicistronic Cables2 knock-in reporter mice that expressed Cables2 tagged with fluorescent reporter was generated. In this model, C-teminal of Cables2 was knocked in with 3xFLAG and 2A-mediated tdTomato. The reporter mice of Cables2 showed the widespread expression of Cables2 in mouse tissues and with high expression in brain, testis and ovary and was reported as useful model and tool for analysing Cables2 function in vivo. Therefore, this reporter mice will be used to examine the Cables2-binding protein in various mouse tissues.
On the other hands, by analysing the Cables2 conventional knock-out, we found that Cables2 also has novel interaction with Rps21, Wnt/beta-catenin and p53 signaling pathways in embryogenesis. These finding will contribute to the comprehensive understanding of Cables2 function in mouse.
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| Strategy for Future Research Activity |
The homozygous floxed Cables2 mice will be mated and propagated with Nanos3-Cre and Gdf9-Cre which plays essential roles in germ cell development. The conditional knock-out Cables2 will be firstly analyzed the phenotype in detail. Then they will be crossed with Nanog-GFP reporter mouse and the embryos will be analyzed during the specification to differentiation of primordial germ cell and germ cell. Moreover, the reporter model will be useful to investigate the Cables2-binding proteins in vivo and the mechanism of how Cables2 regulates Nanog expression and Smad2 transcriptional activation in PGCs and male germ cells will be discovered further.
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Research Products
(2 results)
