More In Situs of Tweety

To continue with the final data analysis for the Tweety paper, I needed to finish up with some final in situ hybridization experiments, histological sectioning, and imaging. In situ hybridization experiments involve creating an mRNA probe that is complementary to a 1 kilobase region of our gene of interest, in our case Tweety homolog 1, Tweety homolog 2, and Tweety homolog 3. Embryos are then permeablized and incubated in the probe so that the probe can then bind to the mRNA to which is is complemntary. A color reaction then allows us to visualize where these transcripts are located through the developing embryo. Performing this procedure on embryos that have been fixed a various stages of development allows us to see the pattern of gene expression throughout embryogenesis. The probes that I was using earlier were consistently robust for Ttyh1 and Ttyh3, but the Ttyh2 probe never had signal. So, I recently relinearized the plasmid DNA containing the Ttyh2 insert and re-synthesized the Ttyh2 probe. Upon doing this there was robust signal for the Ttyh2 gene starting at stage 35. Once this issue was fixed, I had gathered enough in situ data to begin wholemount and histological analysis of my in situed embryos.

Imaging and histology is both a great and hard place to be in the project. While all of the experiments and wet lab work is done, imaging and crysectioning is laborious and time consuming. Imaging will involve setting aside some particularly beautiful embryos and taking pictures of the expression patterning using a microscope attached to a camera. Histology involves embedding embryos in tissue freezing medium and slicing them into 18 miconr thick sections. Both of these require fine motor skills and patience!