Functional investigation of Cables2, a novel transcription cofactor regulating Nanog expression through Smad2 activation, during germ cell development

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K16020
• Research Institution: University of Tsukuba
• Principal Investigator: DINH T.H.TRA 筑波大学, 医学医療系, 研究員 (30816476)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: germ cell / primordial germ cell / embryogenesis / Cables2 / Smad2 / Nanog

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 was recently found as a novel transcription cofactor of Nodal/Smad2 signaling pathways in regulating Nanog expression in mouse gastrulation. Furthermore, Cables2 is broadly 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. Therefore, the temporal function of Cables2 in embryogenesis, specifically in germ cell development should be explored. Moreover, the Cables2-binding protein facilitating Cables2-Smad2/Nanog in primordial germ cells and germ cells will 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 homozygous mice were propagated by intercrossing of heterozygous floxed Cables2 mice. Generally, the physical appearance was normal in both heterozygous and homozygous mice suggesting that Cables2 conditional mice are normal and healthy. The establishment of PGC-like cells from pluripotent stem cells is setting up. In additional, to observe and collect the stable PGC in vivo, the reporter mice of Cables2 were generated and be propagating.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Up to date, there is no commercially valuable Cables2 antibody and expression of Cables2 in vivo remains unclear. Therefore, 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 preliminary data from screening analysis revealed the expression of Cables2 in mouse adult tissues, specially in ovary and testis.

Strategy for Future Research Activity

The homozygous floxed Cables2 mice will be mated with several Cre mouse strains which which plays essential roles in germ cell development. The conditional knock-out Cables2 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. Besides, the Cables2 reporter mice will be analyzed and characterized at early development and adult stage to reveal the expression and cellular localization of Cables2 protein. 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.

Causes of Carryover

In this year, the gene-modified mice were generated and propagated overall. The incurring amount therefore will be saved and used next year to maintain and breeding mouse strains, purchasing reagents, antibodies and experimental kit for screening analysis.

Research Products

• [Presentation] Cables2 regulates mouse gastrulation by stimulating Wnt/beta-catenin signalling pathway (2019)
  • Author(s): Tra Thi Huong Dinh, Hiroyoshi Iseki, Seiya Mizuno, Saori Iijima-Mizuno, Yoko Tanimoto, Yoko Daitoku, Kanako Kato, Masatsugu Ema, Akiyoshi Fukamizu, Mitsuyasu Kato, Satoru Takahashi, Ken-ichi Yagami, Fumihiro Sugiyama
  • Organizer: The 2019 International Symposium of Korean Association for Laboratory Animal Sciences (KALAS)
  • Int'l Joint Research / Invited