Team 6
Introduction
The purpose of this course is to analyze the impacts of known amyotrophic lateral sclerosis (ALS) contributing genes, that influence both neurons and glia, by manipulating their presence within Drosophila (fruit flies). Through the process of introducing disease genes to new fly generations, tracking disease progress through locomotor tests, and collecting other relevant data throughout the aging process of the flies, we are able to understand how certain genes contribute to the onset of ALS and influence the associated symptoms. Overall, this course teaches students how to communicate effectively within a group setting, how to work successfully in a research environment, and contributes to the development of skills essential for future work in the scientific field.
General Information
Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease that impacts the function of motor neurons in the brain and spinal cord. The disease initially impacts the strength and function of voluntary muscles but ultimately progresses to the inability to eat, move, speak, or breathe. Currently, there is no complete cure or exact known cause.
Fly Gender
When analyzing drosophila, making sure you can tell the difference between male and female flies can be essential for success. Males generally have a shorter, more rounded abdomen when compared to the long, pointy abdomen of females. Males also have very dark and visible genitalia that are easily identifiable when examining their abdomen. Males can also be identified by the bristles found on their front legs, "sex comb," but this can be difficult to see.
Virgin Flies
Collecting female virgin flies is an essential aspect of setting up accurate crosses. These females ensure that the cross you manually set up (controlling genes in the process) produces offspring directly from your cross and not from previous random mating. It can be difficult to determine if a female fly is a virgin, but there are some obvious traits to be aware of. These flies will be much larger and lighter in color (sometimes translucent) when compared to their mature counterparts. If found early enough, these female virgins will have a dark spot on their abdomen (last meal prior to pupating). It is important not to mistake virgin males for females. Females will generally be larger and lack a dark genital area at the tip of their abdomen.
Tubes (containing food) flies are maintained in.
Basic drosophila life cycle.
Methods
Locomotor Tests
Procedure:
Before running any locomotor tests ensure that a notecard has been created containing test fly information. (Card must contain fly genotype, birthdate, numbers, date of test, and team number)
Transfer test flies from their food vial into a graduated cylinder marked at the 110 mL line.
Once all flies have been transferred remove any funnels used and block the opening of the cylinder with para-film.
Allow the flies 60 seconds to recover from the transition.
Ensure camera is facing the 110 mL line, click start on the camera then show the notecard.
Hit the cylinder against a soft surface a total of five times in a random pattern to knock the flies to the bottom.
Immediately after this, start a timer for two minutes and let it run.
Once two minutes have passed, stop the recording and transfer flies back into their vials.
Video Analysis
Procedure:
Download locomotor test recordings from the camera and move into box.
Open up analysis spreadsheet and insert information such as strain name, birth date, test date and number of flies.
Watch video of locomotor test. In ten-second intervals record the number of flies that cross 110 mL. (Easier if video is set to 0.5x speed)
Keep track of flies that go back below 110 mL. If so subtract the fly from the others.
Add the collected data into the correct spreadsheet.
Calculate the percentage of the amount of flies with successful climbs.
Collect other information that may be useful e.g. averages, standard deviation.
Compare data collected to previous weeks and trials.
Coding
Coding was a little tricky because we weren't able to find any p-values in our data sets. We discovered that our data was actually written in R and we didn't have the skills to transfer it to Python and then open the data sets.
However, we found a supplemental chart in our article (shown to the left), that showed us which genes were up and down regulated and gave us p-values. We then looked in the fly ortholog set that we were giving and found that only two genes, SOAT1 and HGF, had a fly ortholog. These are the two genes that we are using for our experiments. Both are upregulated in ALS, as seen by the positive log fold change value, and they both have significant p-values.
Genotypes of Parents
Genes of Choice
SOAT1
Fly Ortholog: CG8112
Upregulated in ALS
Function:
- Initiates the formation of fatty acid-cholesterol esters from cholesterol to prevent an over-accumulation of free cholesterol at cellular membranes.
Involved in cholesterol metabolism.
Hypothesis #1
SOAT1 is indicated to be upregulated in ALS, which suggests that there are higher levels of cholesterol esters intracellularly. When there are high levels, SOAT1 comes in and converts them to cholesterol esters in lipid droplets. Higher amounts of CE and LD can cause toxicity in cells which might lead to the symptoms seen in ALS.
Hypothesis #2
Our second hypothesis is that the upregulation of SOAT1 is a response to ALS symptoms rather than an underlying cause of the disease. We read that the acid bile branch of biosynthesis is found to be defective in ALS, this prevents the CNS from removing excess cholesterol. We hypothesis that SOAT1 might be upregulated in attempts to lower the amount of cholesterol in the CNS.
HGF
Fly Ortholog: CG0283
Upregulated in ALS
Function:
In Humans -
Can stimulate growth and division in many cell types making it important for wound healing.
Regulates cell motility and morphogenesis.
Forms new blood vessels (angiogenesis).
In Drosophila -
Involved in proteolysis.
Enables serine-type endopeptidase activity.
Hypothesis:
Upregulation of HGF leads to...
Since HGF can promote the survival of neurons and glial cells it is possible that overexpression of HGF is supporting damaged motor neurons allowing them to survive longer. HGF also promotes the growth of astrocytes which have been shown to lead to the death of healthy motor neurons in ALS.
Results
AlrmGAL4 vs TDP-43 Week 3 Data
We were very confused by this data because the TDP-43 flies (orange line) appear to be doing far better than the normal alrmGAL4 flies (blue line).
AlrmGAL4 vs TDP-43 Week 4 Data
Again, this data was strange to us because the TDP-43 flies appear to be doing even better than they were in week 3, and they are still doing better than the alrmGAL4 flies.
What could be an explanation for these graphs?
The biggest factor that affected these graphs was lack of data.
TDP-43 Week 3 had 7 trials
TDP-43 week 4 had 4 trials
AlrmGAl4 week 3 had 4 trials
AlrmGAL4 week 4 had 3 trials
Because there wasn't very much data, the averages were easily thrown off (see examples).
The same thing can be seen when looking at the data for TDP-43 at week 3. There were 4 trials that had almost 100% of flies climb past the line, which is not what we would expect for week 3 flies with ALS. These 4 groups raised the average number of flies that climbed past the line and is the reason why we see on the week 3 graph that the TDP-43 flies did better than the alrmGAL4 flies.
Why are we seeing inconsistencies in the data?
Possible inconsistencies in how we run our behavior tests/unreliableness of behavior tests.
Behavior tests are not being run at the same temperature/pressure/light every time (maybe the flies just don't like being in the basement).
AlrmGAL4 flies express TDP-43 in glia instead of neurons, maybe expressing TDP-43 in glia is not as effective as expressing it in neurons and that's why the flies aren't showing typical ALS behavior.
Behavior is hard to quantify--the flies are often unpredictable and like to do what they want.
We shouldn't discount our data just because it is the opposite of what we expected.
ANOVA Results
The 4-way comparison ANOVA did not produce a F-ratio that was in the critical region (F >2.41601) for any of the time points. The F-ratio values were super low for all of the time points and none of them even came close to landing in the critical region. I ran the test multiple times and multiple ways, and I always ended up with F-ratio values that were very far off from the critical region.
Why did this happen???
Each group had a different amount of trials and this could have affected how well the ANOVA test was able to compare the four different groups.
The data was kind of off because each group had some trials that were good and some that were bad.
Conclusions
Based on our results, we can't really make a conclusion about the results in the graphs, especially because the ANOVA didn't reveal a significant difference between the groups. The graphs look like there should be a significance between the groups, but the ANOVA test didn't reflect that. Also the graphs presented in the results section demonstrate that the flies that had the ALS gene (TDP43) generally performed better than those that did not (alrmGAL4). This is not what we expected and there are several factors could have possibly contributed to these results, as we explained above.
What Did We Learn?
Although we did make a lot of progress this semester there are procedures that we, and future students, could enforce to ensure that everything goes smoother. Something we definitely have to improve on in the coming semesters is keeping up with our stocks. Although we did label the vials it was not until much later that we figured out a better labeling system to make sure that we flipped our flies on certain days. Practices that we found to really help us work better as a team were utilizing the team notebook and writing down what needs to be done, as well as what has been done in the lab that day. As long as you keep good communication with your team everything should be much less stressful! It is also important to utilize the days of the team meeting in order to organize what everyone will be doing for the rest of the week.
Overall, it is very important to maintain good communication and documentation within a group setting. By implementing all of these previously suggested measures, future groups will benefit and succeed with in the lab environment.
What do we want to teach our incoming students?
Aside from the basic fly information (males vs females, collecting virgins etc.) we want to make sure we educate the new students about the genes we chose and why we chose them. This will ensure that they have a good understanding of what we will be testing in lab.
Explain to them the process of how we set up our crosses so they understand the genetic work that goes into making crosses and deciding what flies you want.
At the end of your lab section, write down a checklist or "To-Do Tomorrow" section in your team notebook. This is especially helpful if you have one or more people in your group that are in the lab by themselves.
Throw out the oldest stock when you flip them, we were not doing this until the very end and it meant that we had like 8 vials from one strain (most of which were too old) and we had to purge a lot of flies.
Example of our team notes that allowed us to plan ahead and work effectively in the lab.
Looking at flies on the fly pad!
Next Steps
The only cross that we were able to get to this semester was the first initial cross of our RNAi genes and the double balancer flies. This means that when we come back from break we still have three crosses we need to do before we are able to run behavior tests on our experimental flies. These crosses will be maintained over the break so the first thing we will do when we come back from break is collect virgins from our RNAi x Double Balancer cross so we can proceed with the next cross (this will be crossing the flies to the double balancer again). We have our crosses all drawn out so we know where we are headed.
Additionally, we need to think about collecting more behavior test data for AlrmGAL4 flies and TDP-43 flies. This is so we can fill in the gaps in our data and ensure that we have an accurate comparison for when we want to compare our RNAi experiment flies to normal AlrmGAL4 flies and normal TDP-43 flies.
Some limitations: lack of locomotor test data, inconsistencies in behavior tests, and time constraints
Our New Year's Resolutions as a group are to be better at taking care of our stocks and to complete our experiment next semester so that we can get results.
By: Jesus, Kamryn, and Isabella
Just For Fun :)
If we asked Reed to check our virgin flies we started calling it having a "gender reveal"
We argued over what color tape we think best represents virgin flies (I say pink but Kelci says red)