Unveiling the molecular mechanism of innate fear using forward genetic screen approach

2016 Fiscal Year Annual Research Report

• Project/Area Number: 15K14874
• Research Institution: University of Tsukuba
• Principal Investigator: Liu Qinghua 筑波大学, 国際統合睡眠医科学研究機構, 教授 (90723792)
• Co-Investigator(Kenkyū-buntansha): 曹 麗琴 筑波大学, 国際統合睡眠医科学研究機構, 助教 (60399475) ; クレウエ・ネベニウス ダニエラ 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (60737667) ; 佐藤 牧人 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (70743699)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: バイオリソース / 先天的恐怖

Outline of Annual Research Achievements

1. To identify core fear genes by forward genetics analysis, we selected 2-methyl-2-thiazoline (2MT), a highly potent analog of TMT, to develop a highly robust odor-evoked innate fear assay by optimizing the age of mice, 2MT dose, and assay duration.
2. Through a dominant genetic screen of ENU-mutagenized mice, we identified a mutant pedigree that exhibited a markedly low freezing rate associated with a high jumping number (>2000 times during 20 min) in response to tFO, hence, was named Popcorn. These behaviors suggest that the Popcorn mutant mice are putative fearful mutants.
3. Popcorn mutant mice are socially submissive as judged by the classical “tube test” and ultrasonic vocal sound in response to female mice.
4. Linkage analysis and subsequent whole-exome sequencing of Popcorn mutants identified the causative point mutation in an intron of Popcorn, an uncharacterized novel gene that is conserved in human, chimpanzee, rat, and zebrafish. This mutation is predicted to result in missplicing. Besides the fearful phenotype, Popm/+ mice exhibited significant subordinate. We hypothesize that Popm/+ mice are prone to accumulation of chronic stress and can be established as models of neuropsychiatric disorders.
5. We recreated the causative mutation in mice by CRISPR to confirm that this mutation indeed causes the “fearful” phenotypes using a battery of innate and learned fear behavior assays. The Popcorn mutant mice will be a valuable model for studying fear-related mental disorders and for developing mechanism-based medicines for mental illness.