Team 2
Manasi Awasthi, Brooke Staples, Divya Maikhuri, and Diya Nath
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Introduction and Purpose
The purpose of our research is to utilize the model of Drosophila melanogaster to learn more about the genes in neurons and glia are involved with symptoms of ALS. Our goal was to see significant effects of differing genes in fly movement and velocity. We planned to analyze this through locomotion assays of the flies that we crossed or had stocks of.
What is ALS?
Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease is a progressive neurodegenerative disease which leads to central nervous system degeneration and its ability to innervate muscle over a relatively short period of time. This affects not only one’s mobility but also the ability to eat or breathe. In 90% of cases there is no family history of the disease where the remaining 10% do have it in their family history. Those with it in their histories have a 50% chance of its onset. Research of the genetic mutations involved could help produce better treatment therapy options for those afflicted.
Model Organism
We chose to use Drosophila melanogaster because they have a relatively fast life cycle which we can observe and produce progeny with. The progeny are produced within 10 days which allows us to collect a greater amount of data. They also have a smaller, but similar genome in comparison to the human genome. Therefore, it is easy to find genes that match between the genomes which lets us have a broader variety of genotypes to utilize. We can also use visible markers and phenotypes to tell what other genes a fly may have that are not easily visible phenotypes. We can also affect their life cycle activity via cryogenics and altering the temperature of their environment.
Differentiation Between Males, Females, and Virgin Females
Differentiating between males and females and finding virgin females are relevant for making crosses. There are a few key differences between males and females. Males (top picture right) have a darker and rounder abdomen and visible genitalia. Females (top picture left) lack such and have no dark spot on the abdomen. They also have a more pointed abdomen than males.
However, we often look for virgin females (bottom picture) to create crosses for our genes of interest. Female virgins are slightly lighter and more yellow than a non-virgin female. They also possess a noticeable dark spot on the ventral side of their abdomen. This is solid evidence that the fly is a virgin female. Make sure to look for the dark abdomen spot that is indicative of a male. You don't want to pick a virgin male by accident!
Genes of Interest
CG5181: This codes for a nucleic acid binding protein which is important for DNA replication, recombination, and repair. This was thought to be more effective on the neural cells when in the context of glial cells.
TDP43: This gene's protein is extremely common in ALS patients and studies. It is clear that it is involved in neural efficacy. It is a DNA binding protein which is found in both familial and sporadic cases. This gene is to be used more as a control than the focus when RNAi genes are involved in the experimentation.
Hypotheses
We hypothesize that if the CG5181 gene plays a significant role in glial health, then its upregulation would produce behavioral effects similar to those caused by the TDP-43 gene.
Methods
Behavior Testing
Locomotion assays via Petri Dish Testing:
We chose the Petri dish test over the graduated cylinder test because it provides more specific and quantifiable data. The procedure involves briefly anesthetizing the flies on ice and observing their movement in a Petri dish. A software program called AnimalTA was used to track their movement and calculate average speed and distance traveled.
Materials Required:
Bucket of ice, Petri dish with lid, test fly vials, funnel, video recorder, paper with genotype label, positioning equipment.
Steps:
1) Put test fly vials into an ice box and wait for 5 minutes until the flies are asleep.
2) Put flies into the petri dish and close the lid. Then place into filming positon.
3) Wait for the flies to wake up fully to avoid skewing the results and measuring their movement inaccurately.
4) Video record the flies’ movement for 2 minutes, showing the paper with the genotype of the tested flies before and after the fly recording.
5) Export and name according to the age, cross date, and genotype.
*Tips: utilize a funnel or two funnels with the thinners ends attched to reduce the risk of flies escaping before or after the locomotion testing.
Data Analysis
Here is a brief tutorial to load and track videos on AnimalTA.
1) Start a “New Project” and name it accordingly.
2) Click “Add Videos” and convert them to the compatible video format.
3) Set all the parameters for the tracking (crop, stabilize, format background, and scale).
4) Flick “Prepare Tracking” and set all the additional parameters.
5) Click “Begin Tracking” and let the video load.
6) Click “Run Analyses” and view your tracked video.
A more in-depth tutorial is linked here: AnimalTA Tutorials
Setting Up Crosses
Over the course of this project, we successfully set up three genetic crosses.
Steps to set up a cross:
Identify and collect the desired number of virgin female and male flies.
Once at least 3 virgin females and 10 males have been collected, transfer them to a fresh food vial to set up the cross.
Flip the vial every 7 days to prevent mixing of parents and progeny.
Results: Control - ALRM GAL4
Data Interpretation:
The data were separated by age in weeks (Week 1, Week 2, Week 3, Week 4, and Week 5). The average velocity for each week's data was calculated through AnimalTA. Each point was graphed to make a scatter plot with error bars to account for any uncertainty or error in a measurement.
These results were used to generate an ANOVA table to test for statistically significant variance.
One-Way ANOVA Table for Velocity
Source DF SS Mean Square F Statistic P-value
Groups (between groups) 4 0.0254 0.006349 0.2523 0.9066
Error (within groups) 40 1.0067 0.02517
Total 44 1.0321 0.02346
Results: Control - ALRM and TDP43
Data Interpretation:
Data was separated by age in weeks (Week 1and Week 4). The average velocity for each week's data was calculated through AnimalTA. Each point was graphed to make a scatter plot with error bars to account for any uncertainty or error in a measurement.
Conclusions, Challenges and Limitations
The overall correlation between the age in weeks and the average velocity of the flies was negative, indicating that the movement of the flies, on average, were slower with age. However, there was not sufficient time in testing to make conclusions. Ideally, we would collect more data of different trials over the course of several weeks. Since we were able to only collect tangible data from week 1 of our gene of interest, we would want to further study the behavior of these flies and do more RNAi testing in future experiments.
Alrm GAL4: We saw the general trend increasing age and decreasing average velocity of the flies.
Alrm and TDP43: We gained enough data to make a graph but insufficient data and the gaps we couldn't fill meant that an ANOVA table couldn't be produced and conclusive statements can't be made at present time.
CG5181: This gene was successfully crossed and week 1 data was produced. However there was insufficient data to create a substantial display.
It was also difficult to get all the data we wanted but we were able to learn a new method of data analysis and gain an understanding of the AnimalTA platform. The main conclusions that we can draw from our experiments would be from our alrm-GAL4 control data. Our p-value was 0.9066 so we cannot reject the null hypothesis. We can see trends on the graph but there is not enough supporting data significant enough to suggest that the age has significant affect. We believe that there was a lot of variation possibly because of temperature affects, light quantity, and possibly timing in the day and week as affecting their behavior.
Next Steps
Now that we have a foundation with the AnimalTA methods and a sufficient organizational system established we are well equipped to run more crosses and test the progeny with the genotypes alrmGAL4 x TDP43 and our CG5181RNAi gene. In the future we need more data in general for these genotypes so that we can compare it to our control and come to make statements that we have confidence in. Our study was limited in terms of time, it was in general difficult to reach the original end goal of our experiments when we had set backs and couldn't extend the time for experimentation into school breaks. We are excited to also possibly investigate other genes in future experiments.
Meet the Team!
References
Bhattacharya, M. (2024). Sourcebook [PDF file]. Retrieved from https://d2l.arizona.edu
What is Als?. The ALS Association. (n.d.). https://www.als.org/understanding-als/what-is-als
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