Jiang Faming, Chen Jiehui, Ma Xirui, Huang Chao, Zhu Shicheng, Wang Fei, Li Li, Luo Lingfei, Ruan Hua, Huang Honghui
Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, The State Key Laboratory Breeding Base of Bioresources and Eco-environments, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China.
Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, The State Key Laboratory Breeding Base of Bioresources and Eco-environments, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China.
Biochem Biophys Res Commun. 2015 May 8;460(3):838-44. doi: 10.1016/j.bbrc.2015.03.119. Epub 2015 Mar 28.
Both the intestine and liver develop from the endoderm, yet little is known how these two digestive organs share and differ in their developmental programs, at the molecular level. A classical forward genetic screen, with no gene bias, is an effective way to address this question by examining the defects of the intestine and liver in obtained mutants to assess mutated genes responsible for the development of either organ or both. We report here such a screen in zebrafish. ENU was used as the mutagen because of its high mutagenic efficiency and no site preference. Embryos were collected at 3.5 dpf for RNA whole mount in situ hybridization with a cocktail probe of the intestine marker ifabp and the liver marker lfabp to check phenotypes and determine their parental heterozygosis. A total of 52 F2 putative mutants were identified, and those with general developmental defects were aborted. To rule out non-inheritable phenotypes caused by high mutation background, F2 putative mutants were outcrossed with wild type fish and a re-screen in F3 generations was performed. After complementation tests between F3 mutants with similar phenotypes originating from the same F2 families, a total of 37 F3 mutant lines originated from 22 F2 families were identified after screening 78 mutagenized genomes. Classification of mutant phenotypes indicated that 31 out of the 37 mutants showed defects in both the intestine and liver. In addition, four "intestine specific mutants" and two "liver specific mutants" showed selectively more severe phenotype in the intestine and liver respectively. These results suggested that the intestine and liver share a substantial number of essential genes during both organs development in zebrafish. Further studies of the mutants are likely to shed more insights into the molecular basis of the digestive system development in the zebrafish and vertebrate.
肠道和肝脏均由内胚层发育而来，但在分子水平上，对于这两个消化器官在发育程序上的异同却知之甚少。经典的正向遗传筛选不偏向特定基因，通过检测获得的突变体中肠道和肝脏的缺陷，来评估负责单个器官或两个器官发育的突变基因，是解决这一问题的有效方法。我们在此报告一项在斑马鱼中的此类筛选。由于ENU具有高诱变效率且无位点偏好，因此被用作诱变剂。在3.5天胚胎期收集胚胎，用于与肠道标记物ifabp和肝脏标记物lfabp的混合探针进行RNA全胚胎原位杂交，以检查表型并确定其亲代杂合性。共鉴定出52个F2代假定突变体，那些具有一般发育缺陷的被舍弃。为排除高突变背景导致的非遗传性表型，将F2代假定突变体与野生型鱼进行杂交，并在F3代进行重新筛选。在对来自同一F2家族的具有相似表型的F3代突变体进行互补测试后，在筛选了78个诱变基因组后，共鉴定出源自22个F2家族的37个F3代突变系。突变体表型分类表明，37个突变体中有31个在肠道和肝脏中均表现出缺陷。此外，四个“肠道特异性突变体”和两个“肝脏特异性突变体”分别在肠道和肝脏中表现出选择性更严重的表型。这些结果表明，在斑马鱼的两个器官发育过程中，肠道和肝脏共享大量必需基因。对这些突变体的进一步研究可能会为斑马鱼和脊椎动物消化系统发育的分子基础提供更多见解。
