Biology research can be a shot in the dark in the early stages.
While this may sound like an “unscientific” approach to research, research begun without a hypothesis can lead in a promising direction and reveal new information. One example is a genetic screen. There are two types of genetic screens, forward and reverse. A reverse genetic screen involves observing the effect of mutating a known gene, moving in reverse from gene to phenotype. A forward genetic screen is a powerful tool for determining what genes are responsible for a known phenotype. Both of these types of genetic screen allow you to elucidate relationships between phenotypes and genotypes that you could never predict. Thus research such as these genetic screens, which are not hypothesis-based, saves the time and effort of making many hypotheses in the wrong direction and also helps to reduce bias. This type of research is extremely useful for the groundwork on complicated biological processes, such as genetic disease.
My research this summer started with a forward genetic screen in the Granato Lab long before I arrived. The Granato Lab at the University of Pennsylvania works with zebrafish, focusing on behavior and development. Although you might not consider fish to be complicated or intelligent animals, zebrafish and other fish can participate in a simple form of learning called habituation. This form of learning is considered “simple” and is seen in most animals, however, the process of habituation is not fully understood which makes it a great candidate for a genetic screen. The Granato Lab sought to understand the basis of habituation in zebrafish by performing a forward genetic screen and investigating promising mutations affecting habituation. The forward genetic screen provided many candidate genes that may be involved in the process of habituation in zebrafish.
In this type of research, some results are more promising than others. One particularly promising gene was the focus of my summer: hip14. There are several reasons this gene was promising for further research. Hip14 is a gene that encodes a palmitoyl transferase that physically interacts with Huntingtin. The relationship between hip14 and Huntingtin points to a connection with learning and with other animals. The protein Huntingtin is produced in many animals, including humans. In humans, Huntingtin protein is implicated in Huntington’s disease, a debilitating genetic disease. The connection between the gene identified in zebrafish and this human illness goes even deeper: zebrafish with mutations of hip14 are unable to habituate to auditory stimuli, and Huntington’s disease patients also have auditory processing deficits.
The hip14 mutant zebrafish. (Fish are surprisingly hard to photograph)
This genetic work was done before the summer, and a line of hip14 heterozygous mutant zebrafish was raised in the lab. Fast-forward to now: my boss, Jessica Nelson, was a visiting professor for SuperLab and reached out to me and another student to run a drug screen for hip14 in zebrafish. Our aim for the summer was to find drugs that restored homozygous mutants’ ability to habituate. Our drug screen was partially hypothesis based. For the beginning part of the internship, Abby and I researched work already done with habituation in zebrafish to identify candidate genes known to be somehow connected to hip14 and therefore affected by a mutation of that gene. We specifically focused on palmitoylation substrates of hip14. Then, we delved further into research to find drugs that were known to affect those candidate genes. Thus we made a short list of drugs that would possibly restore habituation to the mutant zebrafish.
The rest of my summer consisted of testing each of those drugs on homozygous mutant zebrafish larvae. This first involved setting up matings between the heterozygous mutant zebrafish to give us a group of offspring, which were both mutant and non-mutant (siblings). One subset of these mixed offspring would receive the drug, and the other subset would not. Therefore we had four categories: mutants treated with drug, siblings treated with drug, mutant control group, and sibling control group. Then we would test their ability to habituate. Have you ever been to an aquarium and noticed that when you tap on the glass the fish do nothing? The normal response to an auditory stimulus is for a fish to quickly swim away. Zebrafish do this in a very characterized manner, forming a perfect curve as they reflexively contract the muscles on one side of their bodies to swim away. These are called short-latency c-bends. Eventually, after many close-together stimuli, the fish will stop responding this way; they are now habituated. This is measured in the lab by an amplifier producing a tapping sound and a high-speed camera recording the movement of the fish after the sound is made. We can then use tracking software to track many, many fish and test whether they change their behavior in response to the sound over time.
Over the summer we varied the drugs that we used, and the concentrations and treatment times for the drugs, searching for a combination that helped the hip14 mutant zebrafish habituate. This involved data analysis where we graphed their response to the sound over time, watching as to whether the mutants ever stopped responding. We then matched our tracking results with the genotypes of the fish, mutant or sibling, and treatment group, drug or no drug. In this way during testing, tracking, and data analysis, we had only a rough idea of who were mutants and who were siblings. It was only after data analysis that the results were revealed by these categories. I am writing this blog post at the end of testing and analysis for our last candidate drug of the summer. Unfortunately, none of the drugs we tested this summer showed any significant restorative effect for the mutant zebrafish.
Results that go against hypotheses or that show a null hypothesis are often pushed under the rug in discussions about research. Many researchers even shy away from publishing negative results or inconclusive results. This practice leads to the loss of scientific knowledge. In my quest to reflect on my research this summer I have been exploring the merits of failed hypotheses and non-hypothesis-driven research. The beginning of this research was not hypothesis-based and led to a very promising connection between a mutant deficient in habituation and Huntingtin. However, the protein encoded by hip14, which physically interacts with Huntingtin, is involved in a complicated network of other genes and proteins. The drugs that we tested this summer came out of a hypothesis: drugs affecting genes that are palmitoylation substrates of the protein encoded by hip14, the former known to increase habituation, would rescue hip14 mutant’s ability to habituate. This sounds complicated, but the short explanation is that these drugs were meant to be positive controls, they were meant to provide a baseline for more “risky” drugs. We assumed because they were connected with hip14 and were shown to increase habituation that some of them would have a positive effect on habituation during our screen. Our negative results do not necessarily point to a failure in our hypothesis as much as the difficulty of predicting these complicated, interwoven pathways of genes and proteins.
However, these results start to elucidate what is not involved in this habituation pathway in zebrafish. Why did our results go so against previous findings in habituation again and again? Is there a missing link or a completely different pathway that is also involved? These are interesting questions that are raised by research that goes against a hypothesis. More than anything this summer has taught me that no matter how “sure” a hypothesis seems, the results will surprise you.