EXPERIMENTAL FIGURE 6-25 In situ hybridization can detect activity of specific genes in whole and sectioned embryos. The specimen is permeabilized by treatment with detergent and a protease to expose the mRNA to the probe. A DNA or RNA probe, specific for the mRNA of interest, is made with nucleotide analogs containing chemical groups that can be recognized by antibodies. After the permeabilized specimen has been incubated with the probe under conditions that promote hybridization, the excess probe is removed with a series of washes. The specimen is then incubated in a solution containing an antibody that binds to the probe. This antibody is covalently joined to a reporter enzyme (e.g., horseradish peroxidase or alkaline phosphatase) that produces a colored reaction product. After excess antibody has been removed, substrate for the reporter enzyme is added. A colored precipitate forms where the probe has hybridized to the mRNA being detected. (a) A whole mouse embryo at about 10 days of development probed for Sonic hedgehog mRNA. The stain marks the notochord (red arrow), a rod of mesoderm running along the future spinal cord. (b) A cross section of a mouse embryo similar to that in part (a). The dorsal/ventral axis of the neural tube (NT) can be seen, with the Sonic hedgehog–expressing notochord (red arrow) below it and the endoderm (blue arrow) still farther ventral. (c) A whole Drosophila embryo probed for an mRNA produced during trachea development. The repeating pattern of body segments is visible. Anterior (head) is up; ventral is to the left.

[Ljiljana Milenkovic and Matthew P. Scott.]