When I was a little kid, I was a frequent visitor at the Museum of Science and loved looking through pictures in anatomy books. As I have gotten older, this passion for science has never left me, and I have pursued many science-related educational opportunities such as the Rivers School bioethics program this past year. I was so excited about the amazing opportunity to intern at the Burns Laboratory this summer and further explore my scientific interests. During my time working at Boston Children’s Hospital, I learned so much from my mentor, Olivia Weeks P.H.D., whose research project centers on using zebrafish to study congenital heart defects associated with Fetal Alcohol Spectrum Disorders (FASDs).
Fetal Alcohol Syndrome affects between 2% and 5% of the global population. Fetal Alcohol Spectrum Disorders (FASDs) is an umbrella term that includes four distinct types, with Fetal Alcohol Syndrome (FAS) being the most severe. FAS can cause craniofacial abnormalities, behavioral and cognitive changes, and learning disabilities. The heart is among the first organs to develop during pregnancy, and with many women not knowing they are pregnant during the early stages of pregnancy, this often leads to unintentional alcohol exposure to the vulnerable fetal heart. This is one of the main reasons Dr. Weeks focuses on congenital heart defects associated with FASDs.
Zebrafish embryos are an ideal model for research due to their transparent embryos, external reproduction, and similar genome to humans. These characteristics facilitate easy analysis of phenotypes at various developmental stages and easy embryo collection. Zebrafish have two-chambered hearts, which is different from the mammalian four-chambered heart. Their hearts initially form as a cardiac cone, then develop into a linear heart tube, and finally mature into a heart with one ventricle and one atrium. Phenotypic changes caused by alcohol exposure can be observed as underdeveloped heart tubes or hearts, improper heart placement in the body, split hearts, or double ventricles.
Before every experiment, we bred zebrafish and collected their eggs. We also treated embryos with varying concentrations of alcohol. This procedure was very easy to do as the embryos have permeable membranes and would simply absorb the alcohol through the water they were submerged in.
The main objective of my first experiment was to study how exposure to alcohol during early development affects heart formation. We hypothesized that alcohol disrupts heart development, specifically in the early stages. After treatment, we performed in situ which is the process of staining the heart gene purple for more clear observation. After three days of this process, I imaged the embryos under a microscope and took photos. The general trend I saw, that went along with our hypothesis, was that the embryos exposed to alcohol displayed notably distinct cardiac defects compared to those not treated with alcohol. At this early stage, the embryos' hearts are still in the linear heart tube phase and have not yet developed into the mature two-chambered structure. The alcohol-treated embryos fail to migrate toward the midline and fuse into a linear heart tube. On the other hand, the control embryos that were not exposed to alcohol had healthy heart formation. Although the condition of the alcohol-exposed embryos may correct itself as the embryos mature, it can result in lingering cardiac issues throughout life.
Wild type(WT) Heterozygous Homozygous
I identified the genotypes for each imaged embryo and looked for a pattern linking genotype to phenotype. The wild-type fish generally appeared normal, but alcohol exposure worsened the phenotypes overall. The mutants had even more severe heart cone migration defects. These defects were similar to alcohol exposed mutants, evidence to our hypothesis that PDGFRA signaling is required for healthy midline migration of the bilateral cell populations.
PI3K signaling occurs downstream of the PDGFRA receptor, so we hypothesized that alcohol might contribute to congenital heart defects (CHDs) through this pathway. To test this, we experimented with different chemical PI3K modulators, which alter the extent of PI3K signaling. First, we tested to see whether the drug VO-OHpic, a PI3K chemical that activates PI3K signaling rescues the detrimental effects of alcohol on midline migration. I treated embryos with just water (controls), water and 5uM of the drug, 0.75% alcohol with no drug, and 0.75% alcohol and 5uM of VO-OHpic. I observed that the embryos that were not exposed to alcohol had healthy cardiac formation and timeline. I observed the embryos 72 hours post-fertilization (hpf), and what I saw was that the embryos exposed to 0.75% alcohol had disrupted heart formation, which was expected. However, the embryos that were exposed to alcohol and then treated with the drug seemed to have much healthier heart cone fusion, with only a little bit of space between the cardiac precursors. These results proved that the drug did eliminate the negative effects of alcohol exposure with improved PI3K signaling.
Apart from experiments, I dissected adult zebrafish to become more familiar with their anatomy and to practice heart extraction. Under a microscope, I used small scissors to make incisions in the skin and open up the body. I tried to locate every organ while following a picture from a zebrafish anatomy paper. Taking the heart out was definitely the most difficult part of the procedure. Using tweezers, I had to push the ventricle down to reveal the aorta that I was to grab onto to rip the entire heart out. I ripped the heart in half the first few times I tried, but by the end, I was able to successfully remove the entirety of the heart from the fish. This experience strengthened my hand precision and patience.
In addition to bench work, I had the opportunity to participate in a student journal club with other interns, many of them being college undergraduates. The club met weekly, where we read and presented data from original research articles. Initially, these articles were challenging to read, let alone understand, but as time went on I learned strategies on how to tackle difficult scientific literature. By the end, I felt a lot more confident about understanding the topics and interpreting the figures that I was to present.
Further, I had the amazing opportunity to shadow Vassilios Bezzerides, MD, PhD, who is an associate cardiologist and assistant professor of pediatrics at Harvard Medical School. Another intern and I followed him as he did “rounds,” a practice in the hospital where the medical team visits patients as a group to review the patient's status and discuss the next steps. We observed Dr. Bezzerides discharge a patient who had gotten a pacemaker implanted a couple of weeks beforehand. The team listened to the patient’s heart and made sure he was all set to leave before sending him off with his dad who had come to pick him up. The second patient we saw was a five-week-old baby who had constant arrhythmias. There were a lot of doctors in the room monitoring his heart rate, which was at an alarming rate of 245 beats per minute. To slow his heart, doctors iced the baby’s head for around a minute and the baby’s heart rate went down to normal. Low temperatures cause blood vessels and arteries to narrow, lowering the rate of blood flow.
I am honored to have had the opportunity to learn from the incredible people in the Burns Lab. I gained a valuable understanding of what a career in research and medicine looks like. I am extremely grateful to everyone at the Burns Lab, Dr. Weeks, Mr. Schlenker, and everyone at Rivers who made this opportunity possible.
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