Post #1: First Week of Zebrafish Research Complete!

Now that my first week of on-campus research is complete, I have details about my zebrafish research to share and a rough outline of what is next for my investigation of the effect of alcohol on vasculature development.

This past week began with my project advisor, Dr. Jenny Rahn, and I deciding on treatment groups and setting the zebrafish up for breeding. We ended up with 9 treatment groups and 1 control. The groups were divided into wells, each containing embryo rearing solution in which the embryos could grow and eventually hatch. Treatment groups received an added percent ethanol (1.0%, 1.5%, 2.0%) at various hours past fertilization (2 hpf, 4 hpf, and 6 hpf). There were enough embryos to have 16 in each treatment group and the control group. While waiting for each time increment, I completed training modules for zebrafish handling and Dr. Rahn and I brainstormed different methods of placing the fish on slides and photographing them under microscopes. I was able to gain more familiarity with a compound microscope and learn how to use a dissecting microscope and a confocal microscope with the help of my advisor.

Each day following treatment was centered around looking at the developing embryos under the microscope, taking photographs, and writing down observations. I was able to see underdevelopment and deformities in the treated groups as early as 24 hours post-fertilization. These defects became more prevalent 48 hpf and 72 hpf, as I observed embryos with cyclopia, deformed or nonexistent hearts, unusually sized tails, eyes, and heads, a lack of pigmentation, and various sizes of edemas surrounding the yolk area. Finally, at 72 hpf, I measured the heart rate of each treatment group before we fixed the larvae in paraformaldehyde (at this early point in development, they do not have systems for experiencing pain and can be safely euthanized). The fish are then kept in a solution called PBS, which stands for phosphate-buffered saline.

With the first round of experimentation complete, this next week will constitute a more focused treatment of embryos. There will be much fewer treatment groups, as I will be attempting to find a happy medium of percent ethanol where deformities can be observed but the embryos/larvae are not completely compromised (in the first week, some groups were relatively comparable to the control group, while others ended up with only a few surviving larvae by 72 hpf). This week will have the added task of imaging the fixed larvae from week 1 and determining the best way to quantify developmental differences. Blood vessel width, blood cell nuclei count, and blood vessel count are all possibilities due to my vasculature-specific research. The strain of zebrafish I am utilizing has green fluorescent proteins (called GFP) tagging vasculature cells, including blood, vein, and artery cells, so under the dissecting and confocal microscopes it is easy to see all the elements of the vasculature and make necessary measurements using image analysis software. I will also be incorporating the heart rate measurements I recorded as another form of data I can statistically test. The goal is to determine how alcohol affects the development of the vasculature and compare the effects to Fetal Alcohol Spectrum Disorder in humans with both qualitative and quantitative data. There is not a lot of research using zebrafish in this specific area with the specific stain my project advisor and I are using, so a lot of our discoveries will be relatively new. The unknown territory is very exciting!

By the time I post my second update, I will be able to explain how we decided to quantify our data, talk about any major trends observed in week 2, and describe the course of action for week 3. For now, I’ll just keep swimming through literature research and lab work, and see what I find! Stay tuned!