Team 3
Firass, Ily, Arabella, Stephanie, and Collin
Introduction
We are studying Drosophila Melanogaster (fruit flies) to learn more about how various genes utilized in neurons and neuroglia that could possibly increase the ability for ALS become more likely. Our Goal was to determine how our genes of interest affected fly motor function and relate our findings to the ALS human homologue. We did this by setting up genetic crosses, performing locomotion tests, and analyzing specific features of fly movement.
What Is ALS?
ALS stands for Amyotrophic Lateral Sclerosis, and it is also known as Lou Gehrig's disease. ALS is a fatal neurodegenerative disease in which motor neurons are degenerated and destroyed, leaving the afflicted without basic motor abilities, such as walking or even chewing. There is currently no cure for ALS, although the progression of the disease can be hindered by medication and therapy. ALS can be genetic, although this only correlates to 5-10% of all ALS cases. ALS is often developed spontaneously, with no genetic background. In humans, life expectancy is around two to five years; the longest being ten years. To tackle the life-threatening issue, we are studying ALS genes on the Drosophila Melanogaster, a model organism who shares a significant amount of genes with humans. Specifically using virgin males and females to create certain genes in specific progeny with integrity.
Genes of Interest
CAMKII: This gene is responsible for regulating synaptic plasticity, excitability, and cytoskeletal interactions in neurons. It is up-regulated in ALS.
DAB2: This is our main gene of interest since it is an adapter protein coding gene found within growth factor and Ras pathways. Through these pathways, DAB2 is responsible for central nervous system development, endocytosis, and protein binding. It is up-regulated in ALS.
Hypothesis:
Due to the little/incorrect information on CAMKII along with the lack of a cohesive data set, we were unable to make a prediction on what effect the presence of this gene has in regards to ALS.
As DAB2 is an up-regulated gene, we expect that its effects would cause a decrease in fly motor function and exhibit ALS like symptoms.
**NOTES: The research of CAMKII was halted mid-semester due to the discovery of the gene being upregulated rather than down-regulated, making the other information surrounding the gene inaccurate. DAB2 with ALS background was not collected for testing as the genotypes did produce viable flies in Dr. B's lab.**
Model Organism: Dr0sophilia
Drosophilia, also known as the fruit fly, are perfect model organisms for ALS testing.
very similar genetic background to humans
we can often find fly orthologs to match human genes, making them perfect for genetic testing (genome is 60% homologous to humans)
easy to work with due to fast life cycle and are relatively cheap
From Left to Right: Male Drosophila, Female Drosophila, Virgin Female Drosophila (as indicated by dark spot on abdomen)
Methods & Procedures:
Gene selection and finding stocks:
We selected our gene last semester by looking at genes that had a more significant change in ALS, by looking at LOG2 FC and p-value’s.
Statistics of our selected genes:
Volcano Plot of whole class Gene stats, with our Gene DAB2 Highlited.
Crosses:
Crosses Proceedure:
Find a minimum of 3-4 virgin females from your desired genotype.
Find 3-4 healthy males from the second genotype you want to cross.
Put all your male and female flies in one food vial and label the cross.
Once larva emerge, flip adults onto a separate vial to prevent mix-up.
Within 10 days (normally) your new flies should be born.
Maintain vials as needed, Flip to new food every week as new flies will start coming.
Unbiased Fly Tracking - Petri-Dish Assay Proceedure:
Separate test flies into groups of four for testing.
Flip test flies onto a vial containing no food.
Place the vial into an ice box for 2-3 minutes, until knocked-out.
While waiting, place a guard ring (onion) inside a clear petri-dish.
Make sure the petri-dish is clear of scratches and debre to unsure no complications arise which tracking.
Transfer knocked-out flies onto the petri-dish and place the microscope disk as a lid, ensuring the white side is facing down, and secure with tape only at the sides.
Securing the tape only at the sides is key, this is to keep it from obstucting the camera's view of the petri-dish.
Place petri-dish on the microscope holder and let flies rest allowing them to fully recover from being knocked-out.
While waiting, Turn on the lights and aim from a distance to the sides of the petri-dish with a slight angle upwards.
The upward angle is imporant to prevent glaring and reflections off the glossy surface of the petri-dish.
Set up a phone for recording as well as a two minute timer.
On a separate petri dish, place the vial stickers on top, additionally another sticker that shows the test date and the fly age in weeks. This way you have all fly information on the dish.
This information includes, Team number, Vial Birth Date, Genotype, Vial name, Fly count, Test date, and Fly age in weeks. Information also present in video file name.
Start recording the video, at the beginning, flash the labeled petri dish for a couple of seconds.
Once the flies are awake, the label has been flashed. Record the flies for two minutes.
Once recording is complete. Move the petri dish into the ice box until flies are knocked-out again.
In the mean time, re-attach labels onto the food vial.
Once flies are knocked-out, remove the white disk and flip the petri-dish on top of the short funnel, transferring the flies back into its original vial.
Illustration for Petri-dish Assay
Organizational Methods:
To reduce team communication errors and to be able to keep everything in check, we developed and adoped a few methods, these methods were not nessisarily first created by us, but it is what we did and what we thought was nessisary. Below the bulleted list you can find examples of each item.
Vial names.
Vial names are used to identify individual vials to reduce communication errors. Think serial number.
Vial Cabinet.
The Vial cabinet is a spread sheet of all the fly vials we have. It mainly shows The vial name, Genotype, Location of vial, vial type, fly count, date, and a notes section to utilze incase somthing needs to be done with the vial.
Team Notebook standard prompt.
We decided to use a standard (fill out) prompt for each lab section to reduce the chances of missing any important information.
Standardized naming of video files to further reduce mixup.
We adopted a standard (fill out) file names for all the videos when uploaded. [Team Number_Vial Name_Age_Trial Number]
Weekly Team Meetings.
Although it did not go as planned, mainly due to conflicting scheduels, we held team meetings to go over what we need to do the following week, this was supplemented by allocated time during tuesday meetings.
Team Notebook Prompt
Screen shot of our first page of the team notebook showing the entry prompt for each lab section.
Vial Name
Example of how we named the vials.
Analysis Methods:
All methods used in the procedure were analyzed with a program known as AnimalTA by researcher Violette Chiara; this program allowed us to track fly locomotive behavior. Results from the program were then exported into excel/google sheets where statistics like average speed and distance were recorded and made into graphs.
Results
Control Vglut GAL4
Graph contains average speeds over the course of four weeks with error bars indicating the variability of data from each of the three trials. During the first week of trials, flies were not traveling with expected control speeds, lowering our statistical significance as seen below. However, the linear resembling decline of weeks two through four illustrates the decrease in motor function proportional to age.
This graph displays distance traveled across four weeks with data averaged from three trials as indicated by the error bars representing variability. Within the first week of trials, expected distance for the control genotype was not reached by the flies, decreasing the overall statistical significance as seen below. Despite this finding, weeks two through four illustrate a linear resembling decline of showing a proportional decrease in motor function in comparison to age.
Vglut GAL4 Average Speed
Vglut GAL4 Average Distance
From the Vglut GAL4 graphs, we had seen proportionate decreases in motor function seen throughout weeks two, three, and four, but week one's variability in average speed and distance had caused our p-value to be higher than the 0.05 and lower range for statistical significance.
DAB2
Due issues with AnimalTA for our second week of DAB2 trials, we can only present the first week of analyzed graphs for both the average distance and distance of the genotype. Comparing these graphs to the Vglut GAL4 fly trials, we can see that the data does display a decrease in motor function. As DAB2 is up-regulated in Drosophila we can predict that these values will follow a linear decline as the flies continue to age.
Additional Graphs Displaying Separated Trials
These graphs were initially created to depict the control genotype Vglut GAL4, through both average speed and total distance traveled covering four weeks with separate trials, as indicated by the different colored data points: Trial 1 (T1), Trial 2 (T2), and Trial 3 (T3). Upon review and calculation of statistical data, it was decided that compiling the trials into the graphs above to receive true averages and accurate statistics through ANOVA.
Conclusions
DAB2
Due to complications with testing our first gene of interest (CAMKII), we were unable to get far enough along on progress with DAB2 to make any certain conclusions. We behavior tested our DAB2 flies in petri dishes for week one, but weren't able to get AnimalTA to cooperate with our second week data, leading to an inability to draw a reliable conclusion. However, we are aware that a reason we are not able to analyze DAB2 flies with an ALS background currently is because they have been dying at higher rates than expected, as well as the small chance of them being able to have the specific genotype we wanted. We were able to determine a common pattern among our control VglutGAL4 flies that over four weeks, locomotive behavior steadily declines.
Further Discussion/Next Steps
As we are aware that DAB2 functions as an adapter protein and as a negative immune regulator, we expected DAB2 to also maybe have a supportive role in controlling anti-inflammatory responses in ALS. Since we did not get to a point of being able to identify whether it actually does this/has other effects when present in ALS, we feel the next step is to carry out the remaining crosses we had planned and weren't able to complete. (shown in an image below).
1) Complete Cross with VglutGAL4/Cyo;Ly/TM6b X IF/Cyo;UAS-TDP43/TM6b,Tbsb.
2) Complete Cross with VglutGAL4/Cyo ; UAS-TDP43/TM6b X +/+; UAS-Dab2 RNAi/UAS-Dab2 RNAi.
3) Analyze progeny with petri behavior testing for weeks 1-4.
4) Use Animal TA to get data and graph.
5) compare to control.
We'd like to test these crosses next Spring semester...
Limitations of the Study:
1) Generalizability: Findings of this study are specifically about and based on Drosophila Melanogaster. While they share a significant amount of ther genome with us humans, it is not certain whether these findings are directly applicable to humans. More testing is required in order to generalize these results for ALS patients.
2) Contributing Factors: Though our results show a steady decline in locomotive function among the control flies, the decline may be attributed to more than just age, perhaps close quarters and other factors play a role in the degradation of locomotion.
3) Epigenetics: As the flies are kept in what may be considered a stressful environment (especially those in vials packed with flies), perhaps the expression of genes is worsened in our lab flies than they'd be in wild flies or in humans leading otherwise stress-free lives with ALS.
Limitations to Completion:
1) Time constraints/Meeting times: Once we actually got to focus in on DAB2, the clock was ticking on this course. We only got to test our DAB2 progeny at week 1 and week 2, and only got AnimalTA compatible for week 1. Additionally, one hour in the lab ended up not being sufficient to communicate successfully within the group so we added another meeting time to sort out important tasks and upcoming issues to worry about. With a year-long course, we feel we could have gotten closer to meaningful conclusions. We would have liked to meet much more throughout the semester and, due to our respective school schedules, found it difficult to do so. Though, we set up extra meeting times, some days it did not pan out leading to tiny setbacks in awareness of information.
2) No second genotype: While many groups were able to focus on two different genes, or have two different possible genes to work on, our experience with CAMKII being a dud left us with essentially useless CAMKII info, and only DAB2 left to test late into the course, minimizing the information we were able to conclude/draw from.
3) Petri dish/Animal TA: Trial and error with petri dish testing caused us to lose some flies here and there. A lot of our videos would come out unsuccessful with AnimalTA due to smudging or scratches that could interfere with the analysis. This would often cause us setbacks in data and the need to redo behavior tests.
