Xing Chaofan, Li Guang, Liu Xian, Deng Xi, Wang Yiquan
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiangan District, Xiamen, Fujian, China.
Int J Dev Biol. 2017;61(10-11-12):785-792. doi: 10.1387/ijdb.170210yw.
Amphioxus is a good proxy for studying the evolution and development of mechanisms during the invertebrate to vertebrate transition. However, one of the major limitations of amphioxus as a model organism is the lack of well-developed technical approaches. Promoters of genes encoding heat shock proteins are useful tools for gene function studies. In the present study, we tested heat shock temperatures on 4-cell, 128-cell, late blastula, mid-gastrula and late gastrula amphioxus embryos and optimized the heat shock conditions. We then examined the expression profile of the endogenous Hsp70 gene in embryos heat-shocked under optimum conditions. RT-qPCR revealed that the expression of Hsp70 could be induced in embryos by heat shock after the 128-cell stage, and in situ hybridization showed that the induced Hsp70 expression was predominantly detected in epidermal ectoderm. Further, we constructed two reporter gene expression plasmids carrying the Hsp70 promoter to evaluate its efficiency in vivo. Microinjection experiments showed strong red fluorescence or LacZ staining signals in injected embryos after heat shock treatment. In contrast to endogenous Hsp70, reporter gene expression was found in all three germ layers at equivalent levels. Finally, using the Hsp70 promoter, we conducted a preliminary functional analysis of two amphioxus genes, Vg1 and Cer, which play essential roles in left-right patterning in vertebrates. After heat shock at late blastula, mid-gastrula or early neurula stages, ectopic expression of Vg1 was detected in each group of treated embryos. Phenotype observation at 3-gill-slit larvae stage showed that ectopic Vg1 expression led to two-left-side or left-right reversal phenotypes, and that of Cer caused a two-right-side phenotype. Our study establishes a thermo-inducible gene expression system which will be a useful tool for gene function investigation in amphioxus embryos.
文昌鱼是研究无脊椎动物到脊椎动物过渡过程中机制的进化与发育的良好代表。然而，文昌鱼作为模式生物的主要局限之一是缺乏完善的技术方法。编码热休克蛋白的基因启动子是基因功能研究的有用工具。在本研究中，我们对4细胞期、128细胞期、囊胚后期、原肠胚中期和原肠胚后期的文昌鱼胚胎进行了热休克温度测试，并优化了热休克条件。然后，我们检测了在最佳条件下热休克处理的胚胎中内源性Hsp70基因的表达谱。RT-qPCR显示，128细胞期之后热休克可诱导胚胎中Hsp70的表达，原位杂交表明，诱导的Hsp70表达主要在表皮外胚层中检测到。此外，我们构建了两个携带Hsp70启动子的报告基因表达质粒，以评估其在体内的效率。显微注射实验显示，热休克处理后，注射的胚胎中出现强烈的红色荧光或LacZ染色信号。与内源性Hsp70不同，报告基因在所有三个胚层中的表达水平相当。最后，利用Hsp70启动子，我们对文昌鱼的两个基因Vg1和Cer进行了初步功能分析，这两个基因在脊椎动物的左右模式形成中起重要作用。在囊胚后期、原肠胚中期或神经胚早期进行热休克后，在每组处理的胚胎中均检测到Vg1的异位表达。在3鳃裂幼虫期观察表型发现，Vg1的异位表达导致双侧左侧或左右反转表型，而Cer的异位表达导致双侧右侧表型。我们的研究建立了一个热诱导基因表达系统，这将是文昌鱼胚胎基因功能研究的有用工具。
