Since I was a child, I have always wanted to go into medicine. Now that I have spent time in the Burns Lab, however, I may want to go into research instead! During my time in the Burns Lab at Boston Children’s Hospital, I learned so much from my mentor, Olivia Weeks Ph.D., whose project focuses on using zebrafish as a model for understanding congenital heart defects in Fetal Alcohol Spectrum Disorders (FASDs). Dr. Weeks gave me an aspect of her project that she had never explored, and I had the opportunity to perform the experiments.
Fetal alcohol syndrome affects 2%-5% of the world's population. FASD is an umbrella term and there are four different types of FASDs with Fetal Alcohol Syndrome (FAS) being the most severe. FAS can result in facial deformities, behavioral and cognitive issues, and learning disabilities. The heart is one of the first organs to develop during pregnancy and that, combined with the fact that most women do not know that they are pregnant until weeks after, leads to unintentional alcohol exposure to a developing and vulnerable heart. This is why Dr. Weeks focuses on congenital heart defects.
Zebrafish embryos are an excellent model for research because their embryos are transparent, they reproduce externally as eggs, and they develop very quickly. This allows for easy analysis of phenotypes at any stage of development and easy collection of embryos. Zebrafish have two-chambered hearts instead of the four that mammals have, however, their genome is strikingly similar to ours. Their hearts start as a cardiac cone before developing into a linear heart tube until eventually forming the mature, two chambered heart. Phenotypic changes due to alcohol can be observed as underdeveloped heart tubes or hearts, incorrect placement of the heart in the body, split hearts, or double ventricles.
The primary goal of my experiment was to understand the impact of embryonic alcohol exposure on heart development. Our hypothesis was that alcohol disrupts heart development. More specifically, we are focusing on the pdgfra gene. This gene has been shown to affect craniofacial development when interacting with ethanol so we are exploring the role of pdgf signaling in ethanol-induced congenital heart defects. Our hypothesis is that pdgfra mutants are more susceptible to alcohol induced CHDs through PI3K signaling.
My project was to identify cardiac phenotypes of 6 hour post fertilization embryos derived from breeding a wild type (+/+) with a pdgfra heterozygous (+/-) adult that were treated with varying concentrations of ethanol. This was to simulate how a very early human fetus’ heart would change due to a mothers alcohol intake during the first few weeks of pregnancy. Pdgfra is a growth protein that is responsible for heart development. Based on simple Mendelian genetics, the embryos from the mating should be half heterozygotes (+/-) and half wild type (+/+). Dr. Weeks’ hypothesis was that the heterozygous animals would have more severe heart defects than the wild types when exposed to alcohol.
I first selected pdgfra+/- fish carrying a fluroescen transgene that marks the heart, Tg(myl7:GFP), and put them in a breeding tank based on sex. I identified the females by their larger, whiter bellies, and the males by their pinker, thinner bodies. I used roughly twenty fish for the breeding to ensure that I would have enough viable eggs. Zebrafish breed in the morning, so I placed a divider between the males and females and took it out when I arrived at work. After a few hours, I checked on my tanks and most of them produced eggs. I consolidated the eggs into four petri dishes and filled them with water containing 0% EtOH (control), 0.5% EtOH, 0.75% EtOH, and 1% EtOH. After they were treated with alcohol I let them incubate for six hours. Next, I discarded the unhealthy or dead embryos by looking at them under a microscope. Finally, I euthanized the fish before fixing them in paraformaldehyde so they would be preserved for my experiments.
I performed an in situ hybridization with the goal of dying the heart purple to be able to better visualize the phenotypes. I first incubated the embryos in a probe that recognizes the myl7 gene which is only expressed in the heart. Then, I added an antibody which binds to the probe. Finally, I added a staining buffer that recognizes the antibody and stains the heart a dark purple. Once this experiment was done, I spent time imaging the individual hearts and prepared them for genotyping.
The alcohol treated embryos have very distinct cardiac defects compared to the non alcohol treated embryos. Because the embryos were so young, their hearts are still in the linear heart tube stage and are not yet in the fully developed, two-chambered stage. The alcohol treated ones failed to fuse into the linear heart tube. Normally as the animals age, this will correct itself, but it can lead to lingering cardiac problems.
After I imaged and organized the embryos, I needed to match the phenotypes with the genotypes so I prepared a PCR reaction. The first gel image shows the amplification of the pdgfra gene in each embryo. I then performed a restriction digest to distinguish the wildtype allele from the mutant allele. Hets had a darker top line and WT had a darker bottom line.
I differentiated the genotypes for every embryo I imaged and tried to find a pattern between genotype and phenotype. The wild type fish look normal but alcohol clearly worsens the phenotypes. There was no clear difference between the wild type and pdgfra hets. We think that this outcome occurred because the embryos were exposed to alcohol too early. There were more defects in the 0.75% embryos than expected, which made it hard to differentiate the severity. If we were to perform this experiment again, we would introduce alcohol at ten hours instead of six hours.
Next, I performed a similar experiment, this time testing for 1% alcohol. Unfortunately, of the 36 embryos I collected, only 27% were wild type which made it very hard to confidently determine a phenotypic difference between the wild type and hets. We still believe that hets are more sensitized to alcohol exposure based on previous experiments, however, because we had so few total wild types, we cannot make a strong conclusion from these data.
PI3K signaling is downstream of the pdgfra receptor, therefore, we thought alcohol might act on through PI3K signaling to cause CHDs. For this experiment, we tested whether the LY294002 drug, which is a PI3K antagonist, influences the rate of bi-lobed ventricles in embryos following alcohol exposure. I exposed embryos to combinations of 0.75% EtOH and 7 mM of the PI3K inhibitor and analyzed their cardiac phenotype at 72 hpf. I found that exposure to the PI3K inhibitor or 0.75% ethanol alone caused a small percentage of embryos to show the bi-lobed ventricle phenotype. However, when embryos were exposed to both the inhibitor and alcohol, I observed a statistically significant increase in the percentage of embryos with bi-lobed ventricles. Therefore, we conclude that decreased PI3K signaling makes embryos much more sensitive to alcohol.
Aside from doing bench work, I was also able to participate in a student journal club, shadow cardiologists, and dissect an adult zebrafish heart. The journal club was every week and we read and presented the data from an original research article. They were very difficult to comprehend at first, but I eventually learned how to understand the topics and figures. I had the opportunity to shadow Dr. Bezzerides, a pediatric electrophysiologist and research scientist, for a day, which was a highlight of my experience. I shadowed EP Lab which is a procedure where he images the heart and tries to induce an arrhythmia before ablating the bad circuit using catheters through the femoral vein in the groin. Dr. Bezzerides and his fellow tried to induce the arrhythmia for two hours, but could not do it and therefore could not perform the ablation. After the procedure, he brought me to see his patients who ranged from six months to eighteen years old. He also took me into two open heart surgeries for a few minutes and explained what the surgeons were doing while we watched. In addition, I was tasked with imaging a line of zebrafish called Casper (like Casper the Friendly Ghost) because they lack all pigment cells, so you can see their organs through their skin. We wanted to test whether we could image the heart through the skin to measure cardiac function (ejection fraction) without having to dissect it. Ultimately, the outline was too fuzzy to make an exact measurement, however I got to dissect a heart anyway. Although we anesthetized the fish prior to the dissection, the heart was still pumping even as it was not connected to the body anymore because the heart tissue remained viable for a certain period.
It was a privilege to be able to learn from so many impressive people in the Burns Lab and I got an excellent understanding of what a career in research and medicine would look like. This experience was truly invaluable and I would like to thank the Burns, Dr. Weeks, and everyone else who helped me along the way, as well as Mr. Schlenker and all at Rivers who made this possible.