Willer G B, Lee V M, Gregg R G, Link B A
University of Louisville, Louisville, Kentucky 40202, USA.
Genetics. 2005 Aug;170(4):1827-37. doi: 10.1534/genetics.105.041608. Epub 2005 Jun 3.
The zebrafish perplexed mutation disrupts cell proliferation and differentiation during retinal development. In addition, growth and morphogenesis of the tectum, jaw, and pectoral fins are also affected. Positional cloning was used to identify a mutation in the carbamoyl-phosphate synthetase2-aspartate transcarbamylase-dihydroorotase (cad) gene as possibly causative of the perplexed mutation and this was confirmed by gene knockdown and pyrimidine rescue experiments. CAD is required for de novo biosynthesis of pyrimidines that are required for DNA, RNA, and UDP-dependent protein glycosylation. Developmental studies of several vertebrate species showed high levels of cad expression in tissues where mutant phenotypes were observed. Confocal time-lapse analysis of perplexed retinal cells in vivo showed a near doubling of the cell cycle period length. We also compared the perplexed mutation with mutations that affect either DNA synthesis or UDP-dependent protein glycosylation. Cumulatively, our results suggest an essential role for CAD in facilitating proliferation and differentiation events in a tissue-specific manner during vertebrate development. Both de novo DNA synthesis and UDP-dependent protein glycosylation are important for the perplexed phenotypes.
斑马鱼的“困惑”突变在视网膜发育过程中破坏细胞增殖和分化。此外，中脑顶盖、颌骨和胸鳍的生长及形态发生也受到影响。采用定位克隆法鉴定出氨甲酰磷酸合成酶2 - 天冬氨酸转氨甲酰酶 - 二氢乳清酸酶（cad）基因中的一个突变可能是“困惑”突变的病因，基因敲低和嘧啶拯救实验证实了这一点。CAD是DNA、RNA和UDP依赖性蛋白糖基化所需嘧啶从头生物合成所必需的。对几种脊椎动物物种的发育研究表明，在观察到突变表型的组织中cad表达水平很高。对体内“困惑”视网膜细胞进行共聚焦延时分析显示细胞周期时长几乎翻倍。我们还将“困惑”突变与影响DNA合成或UDP依赖性蛋白糖基化的突变进行了比较。综合来看，我们的结果表明CAD在脊椎动物发育过程中以组织特异性方式促进增殖和分化事件方面起着至关重要的作用。从头DNA合成和UDP依赖性蛋白糖基化对“困惑”表型都很重要。
