Author: Chloe Kuebler Major: Biology

During the fall semester of 2020, I continued my project with Dr. Paré’s developmental biology lab to define the molecular mechanism behind embryo compartmentalization in Tribolium castaneum. My goal was to use RNA interference to disrupt the function of the Ten-m protein associated with embryo compartment boundaries in the flour beetles. Dr. Paré’s work with Drosophila suggests that teneurin proteins such as Ten-m play a role in cell polarization during compartment boundary formation. Defining this system in Tribolium would help establish the evolutionary history of insect development between slow-embryogenesis beetles and rapid-embryogenesis flies.

In order to generate Ten-m knockdown Tribolium embryos, I used PCR and SP6 reverse transcriptase to generate double-stranded RNA matching the Ten-m sequence. This process required started with the isolation of genomic Tribolium DNA last semester, which was ultimately trickier than anticipated given that I was using the wrong primers and trying to target a sequence several thousand base pairs long. After some troubleshooting with my mentor, we were able to generate new primers that allowed us to isolate and amplify the Ten-m gene from Tribolium embryos. The Ten-m PCR product was then reverse transcribed to generate double-stranded RNA. This semester I followed the same process to create double-stranded RNA for another gene, Caudal (CAD), as a second positive control. After I successfully made the double-stranded RNA to knockdown both Ten-m and CAD, I started focusing on developing the injection procedures for the adult beetles. With the help of Dr. Tim Evans, Dr. Paré and I were able to set up a system for injecting adult female beetles using a small air pump and glass needles pulled from capillary tubes.

We started the first round of injections in October of 2020 and began with the negative control group. For this set of injections, we used sterile water and dye to mimic the injection conditions without using the double-stranded RNA. Beetle injections proved to be far trickier than I originally imagined. While the process of actually injecting the beetles was straightforward, getting the beetles to survive the process was the real challenge. The first three rounds of injections resulted in 0% viability. I spent the rest of the semester altering injection conditions and aftercare to prevent the death of the injected beetles. At the end of the semester, Dr. Paré identified that one of the biggest issues was likely the shock of adding pure water and disrupting the natural electrolytes of the beetles. In the last round of injections, I performed before the end of the semester, I included phosphate buffer solution in the injection mixture which resulted in the first viable injected females. Next semester, I will continue injections for the negative control and begin injecting for the positive control groups to knockdown Ten-m so the resultant phenotype can be observed using confocal microscopy and antibody staining.

This project was one that Dr. Paré had in mind when I began working in his lab in 2018. I had taken one of Dr. Paré’s courses and I knew he was looking for undergrads, so I approached him about working in his lab. Based on my background in both biology and anthropology, I wanted to work on a project related to the evolution of development. Given the COVID situation, this semester did not go entirely as planned. My time spent in the lab was fairly limited and presented many challenges. Dr. Paré was eager to help me with my project, but we were not able to meet very frequently given our busy schedules and limits on how many people could be in the lab at one time. The virus also prevented any presentations at conferences given that everyone was behind schedule from not being in the lab during the spring or summer, and travel was not feasible.

This research has the potential to help identify the role of Ten-m protein in the process of embryo compartmentalization. The results of this research can then be compared to studies using Drosophila to determine whether the mechanism behind compartment boundary formation is evolutionarily conserved If this mechanism is not conserved, this will show that the molecules that control boundary formation can change over evolutionary time. The results of this study will provide developmental biologists like Dr. Paré with a more comprehensive understanding of how teneurins function to control tissue structure at a molecular level, which could have important ramifications on research into birth defects, neurobiology, and cancer. This research will also improve my understanding of the research techniques I will be utilizing in graduate school.