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.

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. 

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.**

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.

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.

  1. 1. 1. 1. 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.

  1. 1. 1. 1. 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.

  1. 1. 1. 1. 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.

  1. 1. 1. 1. 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.

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.

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.

Control Vglut GAL4

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.

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.