Abstract The advent of molecular biology has led to the identification of many genes with potential roles in the development of the mammalian embryo. These include homologues of Drosophila developmental genes; Multigene families of growth factors, receptors, kinases, and transcription factors; proto-oncogenes; and genes mapping to spontaneous or induced mutations. An important first step in the characterization of these genes is analysis of their pattern of developmental expression, particularly since this can yield clues as to their function. There are two major approaches used to determine sites of gene expression: the detection of protein by immunocytochemistry (see Chapter 7), and the detection of RNA by in situ hybridization. In situ hybridization takes advantage of the specific annealing that occurs between singlestranded nucleic acids of complementary sequence. The presence of a specific mRNA (designated by convention as sense-strand RNA) is revealed by using a labelled antisense probe of complementary sequence to hybridize a fixed tissue section. A major advantage of the in situ hybridization technique is the relative ease and speed with which specific probes can be produced from genomic or cDNA clones, or by the synthesis of oligonucleotides. However, determination of the sites of protein accumulation is also necessary in the longer term, in part because of the possibility that translational control occurs, and also since knowledge of the cellular location of the protein is important for further analysis of its developmental role.

Wilkinson, D. G., & Green, J. (1990). In situ hybridization and the three-dimensional reconstrt1ction of serial sections. Postiplantation Mammalian Embryos, 155–172.