In situ hybridization (ISH) methods remain the most popular approach for profiling the expression of a gene at high spatial resolution and have been broadly used to address many biological questions. One compelling application is in the field of evo-devo, where comparing gene expression patterns has offered insight into how vertebrate development has evolved. Gene expression profiling in the invertebrate chordate amphioxus (cephalochordate) has been particularly instrumental in this context: its key phylogenetic position as sister group to all other chordates makes it an ideal model system to compare with vertebrates and for reconstructing the ancestral condition of our phylum. However, while ISH methods have been developed extensively in vertebrate model systems to fluorescently detect the expression of multiple genes simultaneously at a cellular and subcellular resolution, amphioxus gene expression profiling is still based on single-gene nonfluorescent chromogenic methods, whose spatial resolution is often compromised by diffusion of the chromogenic product. This represents a serious limitation for reconciling gene expression dynamics between amphioxus and vertebrates and for molecularly identifying cell types, defined by their combinatorial code of gene expression, that may have played pivotal roles in evolutionary innovation. Herein we overcome these problems by describing a new protocol for application of the third-generation hybridization chain reaction (HCR) to the amphioxus, which permits fluorescent, multiplex, and quantitative detection of gene expression in situ, within the changing morphology of the developing embryo, and in adult tissues. A detailed protocol is herein provided for whole-mount preparations of embryos and vibratome sections of adult tissues.
Keywords: Amphioxus; Fluorophore-labeled; Gene expression profiling; HCR; In situ hybridization; Multiplex; Single-cell resolution.