Summary: In situ hybridization is a key technique in molecular biology for analyzing gene expression patterns in zebrafish embryos. It is particularly useful for identifying genes with shared expression patterns, known as synexpression groups, which can indicate involvement in the same biological processes or signaling pathways. A limitation of this method is that RNA probes cannot penetrate well beyond the first 2 days post-fertilization, restricting its use to surface tissues in later developmental stages. For deeper tissue analysis, histological sectioning is necessary. Despite this, in situ hybridization remains effective for studying gene expression in endodermal derivatives and sensory organs during late embryogenesis and early larval stages. The RNA probes used can be generated either by in vitro transcription from plasmids or by PCR amplification. The chapter provides detailed protocols for whole-mount RNA in situ hybridization in zebrafish embryos.

Importance: This method is crucial for advancing our understanding of gene function and regulation during zebrafish development. It aids in the discovery of new genes involved in specific developmental processes and enhances our knowledge of embryonic patterning.

Contribution to Literature: The chapter contributes by offering a comprehensive guide for researchers to study gene expression in zebrafish, a model organism widely used in developmental biology and genetics.