Amphioxus Whole-Mount In Situ Hybridization

INTRODUCTION

The in situ hybridization (ISH) technique is one of the basic methods of developmental biology. It allows the distributions of specific transcripts to be detected in fixed cells or embryos during development. This protocol describes ISH of digoxigenin (DIG)-labeled antisense RNA probes to whole-mount amphioxus embryos and larvae. Embryos are fixed and permeabilized before being hybridized with the DIG-labeled antisense RNA probes. Following hybridization, transcripts are detected immunohistochemically with an anti-DIG antibody conjugated to alkaline phosphatase. The gene expression patterns are visualized in the embryos with colorimeric substrates of alkaline phosphatase. Labeled embryos are photographed as whole mounts, after which they can be embedded in Spurr’s resin and sectioned if desired. To facilitate the handling of small amphioxus embryos during whole-mount ISH, we developed a method of solution exchange using homemade mesh-bottom baskets and multiwell plates. This method speeds up time-consuming aspiration and decanting of buffers and avoids accidental loss of precious embryos during these steps. Because ISH conditions are the same for most of the probes tested, this design allows high-throughput analysis of gene expression during embryogenesis to be carried out.