Yao Likun, Chen Jing, Wu Xiaotong, Jia Shunji, Meng Anming
Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Hum Mol Genet. 2017 Nov 1;26(21):4168-4180. doi: 10.1093/hmg/ddx305.
Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients.
细胞分裂周期6（Cdc6）是真核生物中在DNA复制起点形成的复制前复合体（preRC）的一个组成部分。人类preRC的ORC1、ORC4、ORC6、CDT1或CDC6中的隐性突变会导致迈耶-戈林综合征（MGS），其特征为出生后生长受损、身材矮小和小头畸形。然而，尚未有MGS的脊椎动物模型报道。通过N-乙基-N-亚硝基脲诱变和Cas9基因敲除，我们在斑马鱼中产生了多个cdc6突变品系。cdc6的功能丧失突变，如cdc6tsu4305和cdc6tsu7cd突变体所表现的，由于细胞周期在S期停滞和广泛凋亡导致胚胎致死。cdc6低表达突变体cdc6tsu21cd的纯合胚胎在胚胎发育过程中正常发育。后来，与它们的野生型（WT）同胞相比，cdc6tsu21cd突变鱼生长迟缓，成年后的体重和体长大大降低，这与人类MGS相似。令人惊讶的是，cdc6tsu21cd突变鱼变成寿命短的雄性，并且无法与WT雌性交配，表明繁殖存在缺陷。在斑马鱼cdc6tsu4305突变胚胎中过表达模拟在一名MGS患者中发现的人类CDC6（T323R）突变的Cdc6突变形式，部分抑制了细胞死亡表型，表明人类CDC6（T323R）突变是一种低表达突变。cdc6tsu21cd突变鱼将有助于检测更多的组织缺陷并为MGS患者制定医疗策略。
