Spring 2022 Group 2B

ALS in Glia

Introduction

Methods

Crosses

Drosophila Lifecycle

Grape Agar Plate Setup

Cross Setup on Plates

Introduction

How does manipulating the expression of ALS genes in the glia cells of fruit flies affect the climbing ability of the flies?

This was the original question that was tested in previous semesters. However, we discovered that the flies we wanted (repoGAL4/UAS-GR80) for testing were dying. So we started this semester with a new question.

At what stage are those flies (repoGAL4/UAS-GR80) dying?

We decided to test this by doing a lifespan viability study using GFP as a biomarker. So to look at the lifespan, we set up crosses where we can collect embryos on grape plates. Then we looked at the grape plates under a GFP filter each day, counting the amount of GFP positive and GFP negative larvae and noting the stage. Using those numbers that we collect, we can determine at what day or stage the GFP negative count decreases. This implies when the flies are dying.

Methods

Crosses

1) Experimental (GR80) Crosses:

a) repoGAL4/TM3serGFP x UAS-GR80/CyO-GFP

So from the above cross, the percentage of GFP negative flies should be 33% to start. Since we know that the flies are dying at a certain stage, this percentage should decrease at some point. Then we can determine at which stage that decrease occurs and do more research into that stage of development to find out what is happening at that stage that may be contributing to their death.

b) repoGAL4/TM3serGFP x UAS-GR80/UAS-GR80

With this cross, the percentage of GFP negative flies should be 50% to start. But the logic should be the same as above.

2) Control (GRcontrol) Crosses:

a) repoGAL4/TM3serGFP x UAS-GRcontrol

We also set up control crosses, where we the percentage of positive and negative flies should hover around 50% and this number should be stable over their lifespan.

Life cycle of Drosophila melanogaster.

Drosophila Lifecycle

Difficulty in finding Drosophila melanogaster at different stages can vary. Eggs and Embryos can usually be found within the yeast paste. In the larvae stage, the new larvae can be different to see. They tend to be clear and thin. The later stage of larvae is the easiest stage to find and transport to a plate. The pupa stage is straightforward but cannot be transported to a new plate.

Grape Agar Plate Setup

Grape agar plates were synthesized by combining the following materials. (Recipe: 40mL grape juice, 60mL H2O, 1g agar, 10g glucose). After measuring out the proper amounts, the materials were melted together in a microwave. Once the materials were melted into a homogenous mixture, they were poured into petri dishes and set aside to cool. We made sure to leave the lids off of the petri dishes until they were fully cooled to avoid issues associated with condensation.

Freshly poured grape agar plates cooling.

Collecting embryos from a drosophila cross on grape agar plates.

Cross Setup on Plates

To collect embryos, first we had to collect parents for the crosses mentioned above. Also, we set up a grape plate with yeast on it. Then we transferred the parents into a cup (shown to the left) with holes poked in the bottom for air, and quickly placed the grape plate over it. The grape plates fit nicely into the cup, but we also placed tape over it to keep the plate from falling out. Then we flipped the cup so the grape plate was on the bottom. We left the parents there to mate for a period of time. The parents were then removed and recollected to be used again in the future. Then we are left with grape plates with embryos on them.

GFP Counting

A GFP biomarker was used to identify larvae with repoGAL4/UAS-GR80. GFP positive larvae were fluorescent and didn't contain our gene of interest and GFP negative larvae were not fluorescent and did contain our gene of interest. The lifespan of GFP negative larvae were measured in an attempt to discover at what stage they were dying.

So each day in lab, we looked at the grape plates under a GFP filter. We transferred any larvae found onto another plate. We noted the stage and if it was GFP positive or GFP negative.

GFP positive and negative larvae under a microscope. A color matching tool was used to compare the colors of the background, GFP negative, and GFP positive larvae. Colors left to right: background, GFP negative, GFP positive

Results

The graph below shows the GFP-Positive Count/Total Count ratio over their lifespan.

Our hypothesis was that the mean would be around 0.50 for GR-cont & GR80/GR80, and 0.67 for GR80/Cyo in the beginning and decrease for the GR80 ones with time.

Conclusion

We are unable to reach any definite conclusion about when flies are dropping dead.

Notes

Reproductive Performance: Low offspring count(~10) for most of our trials except the last one(~30). Difference: it had older males(~14 days). Churchill et al(2019) reports that 'Older males(14d) were less likely to receive rejection behaviours, as were starved males', but don't directly give an age vs age reproductive performance data. Mossman et al(2019) do give age vs age data but they only compare 3d vs 45d. We may look into this in more detail later.
Transferring Larvae: We believe unintentional stabs while transferring larvae and embryo from one grape plate to another are killing them. We shouldn't use anything with sharp ends while dealing with them from now on.
Counting Larvae: It's hard to get accurate count without transferring We should experiment with videotaping like below and then analyzing it with some software.

References

Churchill, E. R., Dytham, C., & Thom, M. D. F. (2019, February 15). Differing effects of age and starvation on reproductive performance in drosophila melanogaster. Retrieved May 2, 2022, from https://www.nature.com/articles/s41598-019-38843-w

Mossman, J. A., Mabeza, R. M. S., Blake, E., Mehta, N., & Rand, D. M. (2019, February 7). Age of both parents influences reproduction and egg dumping behavior in drosophila melanogaster. OUP Academic. Retrieved May 2, 2022, from https://academic.oup.com/jhered/article/110/3/300/5308908