Blog post 3: What’s a double in situ?

The second part of my project dealt with the analysis of the frog embryos after a transplantation experiment. Beyond looking at the obvious physical appearance of the embryos (mentioned in my first post), I wanted to see how transplantation at different stages might affect the expression of several regional marker genes. These are genes that are expressed in specific locations along the anterior-posterior axis in Xenopus laevis. If an embryo does not fully recover from rotation, you might expect to see anterior genes expressed in the posterior, posterior genes expressed in the anterior, or an absence of gene expression.

The goal of an in situ hybridization is to mark the expression of a certain gene in an organism. When a gene is expressed, regions of DNA are transcribed into mRNA (messenger RNA), which can then go on to be translated into various proteins. Although the DNA in every cell in an organism’s body is the same, different parts of that DNA are transcribed into mRNA depending on the time of development and the location in the organism. That’s why your head is different from your feet – and why we see different genes expressed along the anterior-posterior axis of Xenopus laevis. DNA is usually double stranded, but it’s transcribed into mRNA that’s single stranded. We can synthesize our own strand of RNA that’s complementary to the mRNA that’s normally present in the frogs (called the endogenous mRNA). This synthesized RNA probe will bind to the endogenous mRNA where it’s expressed.

The first day of an in situ involves permeabilizing the embryo so the RNA probe will be able to enter the tissues and cells. At the end of the day, the probe is added and incubates overnight. Day two consists of washes to remove any excess probe. At the end of day two, an antibody (coupled with the enzyme alkaline phosphatase) is added that will bind to the probe. On the third day, excess antibody is washed away. At this point, the endogenous mRNA is bound to the RNA probe which is bound to the antibody which is bound to alkaline phosphatase. Finally, the alkaline phosphatase catalyzes a color reaction, and purple color substrate appears wherever the gene is being expressed.

Since my project is also looking at where the transplanted tissue has incorporated into the host embryo, I need to perform a second round of hybridization. Remember how the donor tissue is marked with a fluorescent dye called fluorescein? We have an anti-fluorescein antibody in lab that will bind to fluorescein wherever it is present. Adding this antibody to the embryos and performing a second color reaction results in a blue color wherever the donor tissue has incorporated.

After this double in situ, there’s still a lot of processing to be done. First, embryos need to be bleached to remove excess pigment – we’re interested in the blue and purple color substrates and this helps us see them better. Then, the embryos are imaged under a microscope. Some of them go on to be embedded in wax and sectioned into very thin slices – this helps see where blue and purple are present in internal structures of the embryo. All of these sections need to be imaged as well, and then it’s possible to analyze where the genes are expressed, if they’re expressed correctly or not, and how the gene expression co-localizes with incorporation of donor neural tissue.

I performed many in situs this summer, and am currently working through the data to analyze it further. It’s exciting to finally have gene expression data after so much work! It was a productive summer in lab and I can’t wait to see what I find out next!