This summer through PURM, I was able to conduct research in Dr. Stephen DiNardo’s laboratory under the supervision of Postdoctoral Fellow, Sara Roberson. The project that I worked on focused on the microenvironment in which stem cells are housed, known as the niche. The niche is responsible for sending molecular factors necessary for regulating stem cell renewal and differentiation. To understand the nature of these microenvironments, the stem cell niche of the Drosophila melanogaster (fruit fly) testis has proven to be advantageous in fulfilling this purpose. The DiNardo laboratory has shown that there are noticeable changes in niche architecture of aged testes. Thus, using the GAL4-UAS system, the laboratory is conducting an RNA interference (RNAi) screen to knockdown factors that may be connected to these age-induced changes. However, this screen requires verification that specific promoters can reliably drive GAL4 in the niche and subsequently knock down the factors. Two promoters, unpaired (upd) and Hedgehog (Hh), are active in niche cells, though their activity is varied.
My project’s goals were to determine which GAL4 should be used for the RNAi screen using green fluorescent protein (GFP) to quantify GAL4 activity. My second objective was to gather preliminary data for the RNAi screen by knocking down one factor, upd, using HhGAL4. For my experiments, I had to cross lines of transgenic fruit flies to construct test animals having the proper combination of genetic components necessary for addressing these goals. This procedure took approximately 22-23 days, after which, I would select from the offspring the proper genotypes containing these components. Then, to image the niches, I would conduct immunoassays, which included performing dissections to extract the testes and immunohistochemistry staining to visualize the niches under the microscope. This step, including imaging the niches, would take approximately 3-4 days. Afterward, I would use Image analysis software, FIJI, to quantify the stained niche cells and GFP+ cells.
During my time in the lab, not only did I learn a multitude of skills, but I also came to understand that science can be not so clear-cut. I was taught certain concepts from my introductory science courses, and so, I assumed that these concepts were well established, yet when I obtained data, oftentimes I would find that these lessons did not always apply, and so, I had to understand perhaps how these exceptions could exist and perhaps develop new procedures for studying them even further. From this, I have gained an appreciation for the scientific method in practice, which I believe will remain with me as I continue my academic career, knowing that the science that I study is always constantly changing.