Team 4

Introduction

Purpose

Our goal for this lab is to study the locomotor effects of genes potentially associated with ALS on Drosophila melanogaster in the hopes of translating our findings to humans.

ALS

Figure 1: A simplified diagram of a healthy nerve cell compared to one affected by ALS. Courtesy of Mayo Clinic News Network, accessed 3 December, 2023.

ALS is a nervous system disease that weakens muscles and affects physical functions. Essentially where a persons brain loses connection with their muscles.

The purpose of our research is to study and understand ALS (amyotrophic lateral sclerosis) genes within Drosophila melanogaster, more commonly known as fruit flies. Our goal is to utilize different genes associated with ALS and study their positive or negative effects on flies.

Drosophila Melanogaster

Fruit flies are used in our study because they have a close genetic background to humans, which allows us access to closely related "in vivo" observations. By studying ALS in flies, we hope to build connections with genes that affect or are affected by ALS in humans.

Drosophila can be studied at different life stages, but we only study them in the adult stage in our research. Because of this, we manage stocks by flipping them into new vials after a certain amount of time to prevent overcrowding and ensure we are testing flies that are the same age.

Our Genes

Our genes were chosen from the Ziff et al. data set. We used Google Co-Lab coding commands to find genes with significant p-values, though we do not know if the p-values provided in the Ziff tables are adjusted p-values or regular p-values.

Hipk

(downregulated in ALS)

signaling pathways like Wingless, Notch, and cell death
Involved in development, proliferation, and patterning of tissues
Associated with cell proliferation and cancer
Modulates expression of CaMKII and PAR-1, which may alter neuromuscular structure/function

TRPM7

(downregulated in ALS)

acts as a cation channel, allowing the passage of various ions, including calcium, magnesium, and other divalent cations.
ALS is connected with low levels of Ca2+ and Mg2+, which create a condition that could affect the proper function of TRPM7 channels.
mutation may play a crucial role in neurotransmitter release in ALS

UBQLN2

(upregulated in ALS)

facilitates delivers of UBQN proteins to proteasome for degradation; enables proteasome binding activity
lack of ability to delivery waste from neurons which leads to toxicity
affects the nucleoplasmic distribution of TDP‐43
overexpression of UBQLN2 → promote the mislocalization of TDP‐43

Methods

Sexing Drosophila

Figure 2: (A) Photo depicting the differences between females and males Drosophila. (B) Photo depicting the difference between adult female and virgin female Drosophila, along with a photo of a supine Drosophila. Courtesy of The Berg Lab, University of Washington, accessed 3 December, 2023.

Distinguishing between males, adult females, and virgin females is a crucial component in conducting our research. To best avoid mistakes in sexing Drosophila, we place them onto CO₂ fly pads to temporarily anesthetize them for a few minutes. During this time, we can use microscopes to sort them into different groups. This process makes it easier for us to set up crosses with adult male and virgin female Drosophila.

Setting up crosses

Our team was responsible for investigating the locomotor behaviors of flies with the alrmGAL4 gene, which is generally expressed in neuroglial cells. Throughout the semester, we set up and tested two crosses:

alrmGAL4 x w1118
alrmGAL4 x UAS-hTDP43

In this process, we had to distinguish between male, female, and virgin female flies. Additionally, we utilized double-balancers to ensure that our flies had the genes we wanted to test. Examples of double-balancers used include: Curly (CyO), Serrated (Ser), Scutoid (ScO), Stubble (TM6bTbSb), and Lyra (Lr).

This semester, we crossed alrmGAL4 flies with UAS-TDP43/TM6C,SbTb flies, which meant that we searched for progeny that did not present stubble, thus ensuring that the flies we plan to test have both alrmGAL4 and UAS-TDP43 genes.

Figure 3: Photo of a wild type Drosophila and one expressing the stubble gene. Courtesy of Basic Science Methods for Clinical Researchers, accessed 3 December, 2023.

Figure 4: Examples of the vials we used to set up throughout the semester.

Behavior testing

Since ALS is a neurodegenerative disease that weakens muscles, observing Drosophila's motor function is an important part of our research.

The procedure for conducting locomotor behavior tests is as follows:

Create a video card that will later be displayed before the beginning of the video that includes: genotype, date of emergence, data of test, number of flies, mortality count, and team number.
Transfer flies from vial into a graduated cylinder with a funnel to prevent any flies from escaping.
Seal the top of the graduated cylinder with parafilm.
Allow flies to rest for one minute. This is to avoid having dizziness as a confounding variable in our test.
Tap down graduated cylinder on the table several times to bring flies down to the base and induce geotaxis.
Observe and film the flies climbing up the graduated cylinder for 2 minutes.
Upload the video for future analysis.

Figure 5: Flies climbing up the graduated cylinder past the 110 mL mark in one of our behavior tests

Data analysis

To analyze the behavior videos, we counted the number of flies that passed the taped 110 mL point on the graduated cylinder. Observations were documented every 10 seconds throughout the 2-minute duration of the video. We are studying the motor capabilities of Drosophila, so flight is not counted in our analysis.

Fly data is then separated by the relative age of flies. Age categories range from Week 1 to Week 4. We used a variety of statistical tests to analyze our data, including ANOVA tests and two-sample t tests.

Results

Our team was responsible for creating graphs based on Week 3 and Week 4 data for alrmGAL4 vs TDP-43 crosses. We conducted ANOVA tests for a 4-way comparison at each time point.

Figure 6: Week 3 flies climbed vs time results from alrmGAL4 x UAS-TDP43 cross

p-value: 0.064806447

mean: 52% climbed

Figure 7: Week 4 flies climbed vs time results from alrmGAL4 x UAS-TDP43 cross

p-value: 0.040811884

mean: 42%

Table 1: Tables summary of ANOVA test results from Week 3 data

Table 2: Table summary of ANOVA test results from Week 4 data

Based on our ANOVA results, Week 3 resulted in a p-value result of 0.0648, which is above our significance threshold of 0.05. Therefore, we do not have enough convincing evidence that there is a significant difference in percentage of flies climbed across the time points.

Week 4 data resulted in a p-value of 0.0408, which is below our significance threshold. Therefore, we have convincing evidence that more flies climb up the graduated cylinder at a later point during the behavior test.

Figure 8: Comparing the percentage of flies climbed over time for Week 3 and Week 4

In comparing the average percentage of flies climbed over time for Weeks 3 and 4 (Figure 3) with a two-sample t-test, we found a p-value of 0.0227, which remarks higher performance for flies that are 3 weeks old than 4 weeks old.

Because Week 2 and Week 3 data points looked similar, we ran a two-sample t-test on Week 1 and Week 4 to search for a significant difference between them. This resulted in a t-statistic of 3.8372 and a p-value extremely close to 0. This means that there is a statistically significant difference between Week 1 and Week 4.

Figure 9: Comparing percentage of flies climbed over time for all four weeks

Conclusion

In running our ANOVA tests, we found that there is a statistically significant difference in the percentage of flies climbed over the course of 120 seconds between Week 1 and Week 4. Younger flies are much more adept at expressing a locomotor response to geotaxis than flies that are four weeks old. This suggests muscles weakening with age.

It remains consistent amongst Week 3 and Week 4 data points that more flies climb up the graduated cylinder at a later point in the behavior test than the beginning. In comparing the two, more flies on average climb up the graduated cylinder when they are 3 weeks old than 4 weeks old.

Future steps

This semester, we did not have sufficient time to investigate the effects of the genes we chose to study. In the future, we hope to be able to delve further into studying our respective genes.

Additionally, we encountered issues with organizing our flipped crosses and keeping track of videos to analyze. Organization of vials is key in this course, so next steps should include establishing a system to clarify which vials should be flipped, tested, or discarded. To ensure everyone receives a well-rounded experience in this course, we suggest keeping close communication with teammates and playing your part in the lab.

Overall, we need to conduct future locomotor behaviors to investigate both the ALS-like phenotype found in UAS-TDP43 and our genes.

References

An introduction to fruit flies. (2015, April 23). The Berg Lab. https://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

Drosophila by david m. Phillips. (n.d.). Fine Art America. Retrieved December 1, 2023, from https://fineartamerica.com/featured/drosophila-david-m-phillips.html

FlyBase gene report: Dmel\Duba. FlyBase. (n.d.). Retrieved from https://flybase.org/reports/FBgn0036180

Madabattula, S. T., Strautman, J. C., Bysice, A. M., O'Sullivan, J. A., Androschuk, A., Rosenfelt, C., Doucet, K.,

Rouleau, G., & Bolduc, F. (2015). Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders. Journal of visualized experiments : JoVE, (100), e52741. https://doi.org/10.3791/52741

Markstein lab | drosophila workers unite! A laboratory manual. (n.d.). Retrieved December 3, 2023, from https://marksteinlab.org/dwu/

Petrucelli, L., & Gitler, A. D. (2017). Unlocking the mystery of als. Scientific American, 316(6), 46–51. https://www.jstor.org/stable/26172693

Sparks, D. (2018, March 14). Learn more about amyotrophic lateral sclerosis, or ALS. Mayo Clinic News Network. https://newsnetwork.mayoclinic.org/discussion/learning-more-about-amyotrophic-lateral-sclerosis-or-als/

Wangler, M. F., & Bellen, H. J. (2017). Chapter 12 - in vivo animal modeling: Drosophila. In M. Jalali, F. Y. L. Saldanha, & M. Jalali (Eds.), Basic Science Methods for Clinical Researchers (pp. 211–234). Academic Press. https://doi.org/10.1016/B978-0-12-803077-6.00012-6