In Situ Hybridization to RNA in Whole Embryos

As indicated in the preceding chapter, few techniques have had such a major impact on progress in the field of developmental biology as in situ hybridization of labeled RNA or DNA probes to detect specific mRNAs in embryonic tissues. Initially, the technique was performed on sections of fixed material. However, this has largely been superseded by the development of techniques for hybridization to RNA in intact embryos. The latter approach involves less effort, and has the additional advantages that a complete temporal study in all tissues can readily be undertaken in one or two experiments and that spatial and temporal changes in gene expression are more readily appreciated ( see , e.g., Fig. 1 A–C,F ). The following procedure is suitable for hybridization to embryos with RNA probes (riboprobes) derived from DNA sequences from the same species and, with slight modification, is frequently suitable for use with probes from other species. This protocol ( 1 – 3 ) is a modified version of that of Wilkinson ( 4 ), and we have included procedures for “two-color” in situ hybridization, which allows the expression of two genes to be examined in the same embryo ( see Fig. 1 F ). Protocols for the sectioning of material subsequent to in situ hybridization ( see Fig. 1 D,E ) are also included.   Fig. 1.  ( A ) In situ hybridization of Fgf-8 to a stage 20 chicken embryo using a mouse cDNA sequence as probe. ( B ) The same probe as in (A) hybridized to a 9.5-d mouse embryo. ( C ) In situ hybridization of a chicken F gf-3 probe to a stage 11 chicken embryo. ( D ) Vibroslice (thick, 50 μm) section of a chicken embryo hybridized with a ret probe showing transcripts in the lateral regions of the otic vesicles. ( E ) Thin (10 μm) section of the specimen in (C), showing Fgf-3 hybridization to the neural tube and branchial pouch endoderm. ( F ) “Two-color” in situ hybridization of Krox-20 (red) and Hox-B1 (blue) showing expression in adjacent segments (rhombomeres) of the embryonic zebrafish hindbrain. ( G ) A typical gel showing DIG-labeled riboprobes before (lane 1) and after (lanes 2 and 3) DNase treatment. Note that the intensities of the probe (lower) bands are much greater than that of the DNA template (upper) bands. Lane M is a DNA mol-wt standard ladder. Note that the RNA in lanes 1–3 appears as a “doublet” even though the enzymes used to linearize the template do not have 3′- overhangs. Whatever the source of the “doublet,” we do not find that it affects the use or sensitivity of such probes. ( See color plate 12 appearing after p. 368.)