3) Characterizing Gene and Protein Expression during Development (by Rey J. Rosa Morales)

Perhaps the most important type of data sought in developmental genetics and evolutionary developmental biology are the expression patterns of specific genes, and the proteins they encode, during development. These patterns are Spatio-Temporal, with a spatial component (referring to specific cells, tissues, segments, or structures) and a temporal component (referring to specific developmental stages). Gene expression patterns can be visualized by three methods, each requiring different tools and hence currently usable for certain species, but not others.

In Situ Hybridization subjects tissues or whole specimens to a chemical process designed to stabilize messenger RNA molecules in the cells in which they are produced (see Figure 2 and the YouTube video). Then a species-specific, single-stranded RNA or DNA "probe" corresponding to the gene of interest is applied. It hybridizes by base-pairing with the mRNA of interest. The probe is either chemically modified so that it can be detected by a staining procedure or labeled with a radioisotope so that it can be detected by autoradiography (Figure 1A). Alternatively, extractions of mRNA from different tissues or developmental stages are run in separate lanes on an electrophoretic gel. The mRNA from the gel is then blotted onto a membrane, which is exposed to the same sort of radioactively or chemically labeled probe used ill the in situ approach. This procedure is known as a Northern blot.

Gene expression patterns can be analyzed at the protein level using antibodies. (The mRNA and protein expression patterns of a given gene may not be identical, due to regulation of translation.) Antibodies are produced by injecting a mammal (e.g., a rat) with the protein of interest (the antigen). The animal produces antibodies (immunoglobulin molecules) that bind specifically to that protein. These "primary" antibodies are collected by passing the animal's blood serum over a resin column containing the antigen and then eluting the antibodies from the column in concentrated form. Tissues or embryos are prepared in a similar way as for in situ staining and incubated with the primary antibody. A secondary antibody, an immunoglobulin that specifically binds to the primary antibody, is then applied to the specimen. The secondary antibody is modified so that it can be defected either by an enzymatic reaction producing a colored product or by fluorescence (Figure 1B). An alternative to staining fixed tissue is to prepare protein extracts from different tissues or developmental stages and run each extract as a separate lane on an electrophoretic gel. The protein from the gel is then blotted onto a membrane, which is then incubated with primary and secondary antibodies as described above. This procedure is known as a Western Blot.

A third method is to study the transcription patterns of particular fragments of putative cis-regulatory DNA by using Reporter Constructs (see the YouTube video) in cultured cells or in transgenic (genetically engineered) individuals. Reporter constructs consist of the regulatory DNA of interest, spliced upstream of a "reporter gene" that encodes a protein whose expression can be easily visualized under the microscope. One such protein is galactosidase, a bacterial enzyme that processes a particular sugar into a blue product. Another is a protein from jellyfish (GFP) that fluoresces bright green when irradiated with light of a particular wavelength. Because reporter construct analysis requires the use of gene transfer technology, it can be undertaken only in certain well-studied model species, such as Drosophila, Caenorhabditis elegans, Arabidopsis, and mice. Figure 1C shows the nematode Caenorhabditis briggsae expressing a GFP reporter construct containing cis-regulatory DNA from the myo-2 gene, which directs the reporter gene's expression in the pharynx.

Figure 1. (A) The probe can be detected by a staining procedure or labeled with a radioisotope, here the probe is detected by autoradiography. (B) The secondary antibody can be defected either by an enzymatic reaction producing a colored product or by fluorescence. (C) Caenorhabditis briggsae expressing a GFP reporter construct. (photo by: A, Matthew Harris; B, photos by John True; C, photo by Eric Haag).

Figure 2. In situ hybridization. Expression of survivin at advanced stages of gut regeneration 
of sea cucumber H. glaberrima (days 12 - 21 after evisceration). (Mashanov and García-Arrarás et al., 2010).

In Situ Hybridization (3min)

Reporter construct (5 min)

References:

1. Gall, J.G; Pardue, M.L. (1969 Jun). "Formation and detection of RNA-DNA hybrid molecules in cytological preparations". Proceedings of the National Academy of Sciences of the United States of America 63 (2): 378-83.
2. Jin, L; Lloyd, RV (1997). "In situ hybridization: methods and applications". Journal of clinical laboratory analysis. 11 (1): 2–9.
3. O'Connor, Clare. "Fluorescence In Situ Hybridization (FISH)". Nature Education.
4. (Link) In Situ Hybridization of RNA and miRNA Probes to cells, CTCs and tissues.