Conquering Conclusions Through Zebrafish Genetic Research

In everyday life, people like to jump to conclusions. For instance, that guy who cut you off on the highway, total jerk; that girl who failed her test, really dumb; and that guy with really long flowy hair under a baseball cap, totally a lax player. With this thinking applied to biological research, that should mean when a genetic screen for a behavioral mutant yields a specific genetic mutation, they’re automatically linked, right? Well, like the prior misguided conclusions, this thought process is way off.

The Big Questions

In biological research conclusions made between mutated genes and various physical outcomes, or phenotypes, cannot be made so quickly. Many biological systems are controlled by more than one gene and sometimes are affected by some other biological function which may also be controlled by various genes. The possibilities are endless in terms of what can really cause these phenotypes. If this is the case, and we don’t get answers immediate from these screens, why do we have them and why are the results deemed so important?

In the case of my research this summer, with Haverford Professor Roshan Jain, the results of these screens allowed us to answer a larger question in biology: How do genes affect behavior?

First off, why do we care? In many cases the “behaviors” which we are talking about in humans are ones which are impairing, like schizophrenia and epilepsy.

So what do we know already that we need to embellish? Well, we (as a scientific community) know that RNA is transcribed from DNA and is then translated into protein. Both on the RNA and protein level, it is understood that cells in neurons and the neural circuits they are a part of are directly affected, depending on the genetic products which were transcribed and translated. Even some of the most minor genetic changes can have a huge effect on behavioral neural circuits. Specifically, the Jain lab and I this summer looked into the acoustic startle circuit and changes which cause learning defects in a specific behavior called habituation.

Ignorance is Bliss

Habituation is a simple form of non-associative learning which occurs when organisms decrease the number of responses or cease to respond to non-consequential stimuli. This type of behavior is very important in terms of research because its implications in survival are critical, and can represent a split second action making the difference between life and death. This summer, we specifically observed this behavior in larval zebrafish (Figure 1) due to ease and accessibility. 

A 30 day old larval zebrafish.

Figure 1. larval zebrafish (age: 30 hrs)

In their larval stage, zebrafish are transparent, making it easy to observe developmental structure as well the general circuitry of neural systems; making observational data simple to collect. Additionally, it is very easy to perform experiments on a large scale using zebrafish as a model organism, allowing mass testing and fewer potential errors due to the size of the experimental pool. Using zebrafish, habituation is also very easy to track on a large scale and can be defined using few factors. 

The habituation mutant which I focused my research on this summer, ignorance is bliss (72FAGA), was identified in a forward genetic screen performed by Professor Jain as well as several members of the Granato lab at the University of Pennsylvania. ignorance is bliss has a mutation of a candidate gene known as ap2s1, a gene controlling the sigma subunit of the AP-2 Complex which targets membrane proteins for endocytosis, a biological trafficking process, and is believed to be necessary in decision making as well as habituation.

Solidifying Findings

Since we can’t jump to conclusions with these findings, a major question which was necessary to address before making any connections/assumptions was “How do we know this specific genetic mutation is what is causing abnormal habituation?” This question was answered using a technique known as a complementation cross. The goal of this test is to cross two organisms, zebrafish here, which have mutations in the candidate gene in question, for our purposes ap2s1. Once the cross is complete, the larval offspring of the cross are genotyped and behaviorally tested to determine whether or not they are mutants for the habituation phenotype and contain two differently mutated ap2s1 alleles from the mutant parents. This is the desired outcome will show that that the specific genetic mutation observed in the genetic screen is truly associated with the mutant habituation phenotype. This is called a failure to complement. A generic outline of this process is shown below (Figure 2).

Figure 2. complementation cross outline

As seen above, the mutant which was generated for the cross to complement with 72FAGA (ignorance is bliss) was an ap2s1-CRISPR mutant, where a targeted insertion/deletion was put into the candidate gene.

Each of these offspring were behaviorally tested using a 16 well plate attached to an amplifier, which repeatedly exposed the larvae to acoustic stimuli, triggering a response. Depending on how the responses changed over time, habituation was calculated. Once behavioral data was collected, the larvae were used as DNA for genotyping. Genotyping revealed whether the tested larvae were heterozygous for one of the mutant ap2s1 alleles, wild type with regular ap2s1 copies, or double mutant trans-heterozygotes for both the CRISPR and 72FAGA ap2s1 genes. This was done through restriction enzyme digest and polymerase chain reaction. The samples were run out on a gel and the bands were observed to make distinctions between larvae. Examples of these digests are seen in Figure 3.

Figure 3. gel results of restriction enzyme digest for 72FAGA and ap2s1-CRISPR mutations – In the left image the restriction site was located in the 72FAGA specific mutation, so the double bars represent the presence of a mutated ap2s1 allele. In the left image, the additional bar represents uncut DNA in CRISPR mutants. The cut site for this specific gel’s DNA was located within the wild type ap2s1 gene, so a CRISPR carrier would have both cut and uncut DNA.

The Results

Figure 4. results of the complementation cross

The graph in Figure 4 shows the combined results from the genotyped larvae and the behavior which was tested prior to genotyping. This was especially exciting because the graph reflects exactly what I wanted to see. While wild type behavior (>50% habituation) was seen in the hets and wild type larvae, mutant behavior (<50% habituation) was seen in some of the 72FAGA mutants (expected as a control) as well as in the trans-hets. This means that the fish failed to complement and ap2s1 is in fact the mutated gene associated with a lack of habituation. With this information at the end of the summer, it is now possible for the Jain lab to raise these fish up for attempted rescue of wild type behavior when they are older, utilizing what we know about them genetically already and our ability to test them.

My Thoughts on the Summer

I know this blog post is fairly dry, but my overall experience with the Jain lab was not. I very much went into this summer thinking that I was done with biology, and this was my last ditch effort to see if I was wrong. I’m a current math major, who frankly has been disenchanted with my experience with biology thus far at Haverford. However, this summer rekindled a lot of the love which I had for the subject. I found myself being very inquisitive in lab because I was genuinely interested in the topics of research and was deeply engaged in figuring out what years’ worth of research would yield. What I found was that there is a tremendous amount of work which goes into research, from asking the big “Why?” question, to getting down into the gritty details, and eventually churning out a result and a publication. Yes, it’s grueling, and yes, it’s challenging, but the results are worth it, and the feeling of pride/accomplishment is unlike anything else. I think that experiencing Professor Jain’s research at the end of its 4-5 year time span really allowed me to be excited by the outcomes of it all, and become more motivated to ask my own big questions. Honestly, figuring things out and realizing that you have accomplished something is one of the most exciting and rewarding events I’ve experienced while in college.

Although I may stay a math major, that doesn’t mean that I won’t keep up my biology studies. In fact, this summer may have even moved me to produce a math bio related senior thesis, and merge my two academic loves: a subject I’ve always loved, and another which I’ve luckily and thankfully become reacquainted with.

One thought on “Conquering Conclusions Through Zebrafish Genetic Research

  1. Thank you Amy . I’m a parent of a little girl with severe autism , a mutation in ap2s1 and fhh3 . She is the most severe case of this mutation and fhh3 . So this research is of great interest to me and gives me information that is hard to come by .

Comments are closed.