Summing it All Up. What an Amazing Summer!

In summary, my summer research project was highly successful in getting a head start on the Tweety research project. While full characterization of the Tweety genes’ expression is still in progress, the ten summer weeks that I spent on the project allowed me to get a thorough idea of the expression and perturbation patterns of the Tweety homolog 1 gene. In the final weeks of the summer, I began to use lacz staining with red-gal to determine the side that received the injection. This entails a similar process of injection to the GFP where I mixed red-gal mRNA with the Tweety mRNA injection mix. However, I no longer to need to side sort the embryos prior to fixing because re-gal staining is a nuclei stain that will last through MEMFA fixing and even an in situ experiment!

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All Aboard the In Situ Train

To continue with my project, I have begun a more in-depth functional characterization of the Tweety Homolog 1 gene throughout various stages of development. To do this, I performed several whole mount in situ hybridizations (wISH) on embryos of various stages and dosages of the TtyH1 mRNA construct. In short, ISH allows me to visualize the expression patterns of specific genes via a purple stain. This four-day protocol was started promptly at 9 AM each morning and was often done in groups for maximum efficiency – hence the title “in situ train.” However, before beginning this exciting procedure, I needed to acquire a variety of experimental and control embryos. To do this, the single-side injected embryos mentioned in my previous post needed to be fixed at various stages. Embryo fixation involves a series of washes and ultimately preserves the embryo at that stage of development.   Once I accumulated a wide variety of stages, (stages 18-35) I was able to begin the wISHes!

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Overexpression of Tweety Homolog 1 Results in Obvious Morphological Defects

Thus far during my research with Dr. Margaret Saha, I have come across some interesting and exciting effects of the experimental procedures previously described in my abstract. After mastering the techniques of microinjections and embryo care, I began routinely injecting the tweety homolog 1 (TtyH1) messenger RNA construct. This entails injecting one cell of a two-cell embryo with an  mRNA construct while leaving the other side unaffected to act as an internal control.  Therefore, Green Flourescent Protein (GFP) is used as a fluorescence marker to indicate which side of the developed embryo received the RNA injection. This procedure involves assembling an injection mix containing 0.25 ng-1.0 ng of the TtyH1 mRNA construct, 0.5 ng of GFP RNA, and Nuclease-free water. In order to have a baseline for the general health and morphology of microinjected embryos, an additional control mix 0.5 ng of GFP is prepared for injection into an external control group.  Embryos are then collected at the two-cell stage approximately 90 minutes post-fertilization and plated for injection. The aforementioned solution is delivered to a single side of the two-cell embryo via a glass needle that is a minute 20 microns in diameter.  Embryos are then kept in a salt solution to be monitored throughout their development. In the days following the injections, embryos are assessed for morphological differences and fixed at various stages in order to have a wide array of ages for the assessment of response to tweety overexpression.  Injections proved to be demanding, all-day events, often resulting in only a few small Petri dishes of embryos. However, once my hands steadied and my technique improved, it became second nature.

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Characterization of the Role of the Tweety Genes in Embryonic Development

While some literature exists reporting that the Tweety gene family encodes for the expression of transmembrane proteins in developing Drosophila, very little work has been done exploring the role of these genes in early vertebrate development. In addition, preliminary lab data indicates that the Tweety gene family plays a role during embryonic neural development of Xenopus laevis or the African clawed frog. To begin this project, the lab has performed in situ hybridizations in Xenopus for these genes which show that the localization of expression of these genes is concentrated in the brain, spinal cord and cranial nerves.  This was followed by the production of misexpression constructs and the synthesis of RNA which will be injected into developing embryos at the two cell stage to influence expression and further understand these genes’ roles in neural embryogenesis. Through manipulating the typical expression of these genes, I plan to ultimately characterize this gene family’s role in application to other developmental mechanisms and explore their function in relation to neuron cell fate. More importantly, the Tweety genes are homologous to genes expressed in humans, and characterization of this gene family could lead to further understanding of human neurological development and disease.