Characterization of an amphioxus heat-shock protein gene promoter and its application in vivo.

Abstract

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.