Brian Wright and Dr. Michael Stark, Physiology & Developmental Biology

The ever-increasing knowledge regarding cellular regulation and programming requires important insights into the biochemical pathways that determine a cell’s fate. For example, the ability to take a stem cell and grow it into a neuron will directly depend on our knowledge of the steps used in the embryo for normal neurogenesis, or the formation of neurons. The ophthalmic placode (a region of thickening cells) of the trigeminal nerve provides a valuable model for studying sensory neurogenesis, since it gives rise exclusively to sensory neurons. This nerve provides tactile sensation to the upper region of the face.

This project aims to elucidate the regulation of Neurogenin2, a basic helix-loop-helix transcription factor that is essential in placode neurogenesis. Thus I set out to study parts of the development pathways of this specific nerve by examining which signals were required to produce the protein Neurogenin2, which serves as an important key for unlocking the development of neurons. As stated above, this information will be important in future research or medical treatments aimed at directing the fate of specific cells. Furthermore, when I selected the project, it was not currently clear as to what signals regulated the expression of this important protein. This project, therefore, was hoped to fill the gap in the scientific community’s understanding of this process.

I studied the development of these cells in chick embryos. The chick embryo offered several advantages over mouse models, such as shorter gestation periods and easier access for manipulating signals sent between cells. I removed the chick embryos from their eggs, detached their heads, and imbedded the heads in collagen tissue in little wells. I then filled the wells with growth serum that contained everything necessary for the embryo cells to continue growing. These steps were performed every time during the experiment.

I then added either DAPT or DMSO (the control). DAPT blocks an intercellular signaling pathway called Notch. This was the ideal choice, because I was trying to determine the functions of Notch signaling in the development of this particular nerve. Since the chemical DAPT is actually dissolved in the solvent DMSO, adding just DMSO served as a negative control. In other words, the addition of just DMSO allowed me to be certain that any changes that I observed were not just due to the heads being removed from the rest of the body or any other potentially important factors.

After incubating the embryos heads for 24 hours, I removed the heads from the collagen. This completed the manipulation of the intercellular signaling pathways. However, I still needed to be able to visualize the results. In order to do this, I used probes that bound to specific mRNA (in situ hybridization) as well as antibodies that bound to specific proteins

(immunohistochemistry). Both of these methods allowed me to locate the targets of interest in order to see if: 1.) the location of these targets was different between embryos incubated in DAPT or DMSO; and 2.) the amount of the targets (either proteins or mRNA of interest) had changed. This process took approximately four days and required me to cut the embryo heads into thin sections that were 1.2 x 10-6 meter thin.
As usually happens in scientific research, this project experienced numerous setbacks and delays as I tried to work through various problems that came up. For a couple of months I was unable to image any of the cells, because the antibodies were not binding to their specific proteins. This was a significant problem since I was unable to visualize the results of my study. Changing some of the solutions and chemicals that I used during the experiment eventually remedied this problem. However, the delay was significant enough to make me worry that I would not complete my project in time!

Eventually, however, I recovered sufficient data to determine that this particular intercellular communication pathway (Notch) does not limit or increase the domain of expression of the protein Neurogenin2. This was interesting, because it was not what I expected. Both Notch and Neurogenin2 are known to play an important role in the ophthalmic trigeminal placode, so I suspected that there might be some deeper connection between the two. Consequently, this raises more questions than were answered, but it propels us further towards understanding the development of this nerve.

It is possible that blocking Notch signaling does result in some variation of Neurogenin2 expression that occurs before the time frame that I looked at. Additionally, it is possible that the level of Neurogenin2 expression was increased, since I did not have any means to quantify its level of expression. This will be an interesting focus of future experiments that aim to further clarify the role of Notch signaling on Neurogenin2 expression within this area of interest.

From this project, I learned about the importance of persevering in research. As I approached my deadline with few results, it would have been easy to give up and not waste my time. However, the closer I got to the deadline, the harder I worked and the more focused I became. As a result, most of my significant results came within the final two weeks of my project (putting me into a mad rush to get all of the data organized and presented!). I am grateful for this pressure and experience, because it helped me to appreciate more fully the success that finally came. I anticipate that I will have many similar experiences in any future research or job opportunities.

Additionally, this project instilled in me an understanding of the importance of continual learning. Throughout this project I had to consult databases and search recent articles for any new information that could help me in my project. For example, I had originally thought that I would look at a different intercellular signaling pathway and for a couple of weeks had planned on pursuing that thought. However, a recent study then came out that answered my question about that pathway and made future plans irrelevant. Thus, no matter what field of science I will be going into, it will be essential to stay updated about new developments and findings.