INRA, UR1037, Laboratory of Fish Physiology and Genomics, Campus de Beaulieu, French National Institute for Agricultural Research, Rennes Cedex, France.
Protim, Inserm U1085, Irset, Campus de Beaulieu, Université de Rennes 1, Proteomics Core Facility, Rennes Cedex, France.
Mol Reprod Dev. 2019 Sep;86(9):1168-1188. doi: 10.1002/mrd.23231. Epub 2019 Aug 5.
Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.
卵生脊椎动物产生多种形式的卵黄蛋白原 (Vtg)，Vtg 是卵黄营养物质的主要来源，但人们对它们在繁殖和发育中的个体贡献知之甚少。本研究利用成簇规律间隔短回文重复序列/CRISPR 相关蛋白 9 (CRISPR/Cas9) 基因组编辑来评估斑马鱼 (Danio rerio) Ⅰ型和 Ⅲ型 Vtg 的必要性和功能。采用多重 CRISPR 方法同时敲除 (KO) 编码所有Ⅰ型 vtgs (vtg1、4、5、6 和 7) 的基因 (vtg1-KO)，以及单独敲除 Ⅲ型 vtg (vtg3) (vtg3-KO)。聚合酶链反应 (PCR) 基因分型和测序、定量 PCR、液相色谱-串联质谱和 Western blot 分析的结果表明，只有 vtg6 和 vtg7 逃脱了 Cas9 编辑。在剩余的Ⅰ型 vtgs 失能的鱼 (vtg1-KO) 中，以及在 vtg3-KO 鱼中，肝和卵中 Vtg7 转录本和蛋白水平显著增加，揭示了一种迄今未知的遗传补偿调节 Vtg 动态平衡的机制。vtg1-KO 雌性产卵数增加了 2 倍以上，而 vtg3-KO 雌性的卵子生育力减半。在仅孵育 8 小时后，vtg3-KO 卵/胚胎的死亡率明显增加，在 vtg1-KO 胚胎孵育 5 天后，死亡率明显增加。来自 vtg 突变母体的胚胎的孵化率和时间明显受损，幼虫出现心包和卵黄囊/腹部水肿和脊柱侧凸，vtg1-KO 幼虫也没有进食和运动活动。到晚期幼虫阶段，vtg 突变要么完全致死 (vtg1-KO)，要么几乎致死 (vtg3-KO)。这些新发现提供了第一个实验证据，证明不同类型的脊椎动物 Vtg 在繁殖和发育过程中的不同时间都是必不可少的，并且具有不同的必需功能。
