Live Cell Imaging of Drosophila Gonads (Update 3)

The end of the summer session is fast approaching and so much has happened since my last post.

First of all, I was finally able to image some differentiating gonads! I found that adding 450 microliters of freshly aliquoted Atlanta Biological’s heat inactive fetal bovine serum (FBS) to my live cell imaging culture media supports cell differentiation. However, I didn’t see much growth of the gonad itself so I am still playing around with the exact formula for the culture media. I recently tried out using Shield and Sang’s M3 medium to isolate gonads and in my culture media in addition to the new FBS. Unfortunately, although there was differentiation of gonads in this culture media, the gonads died about 6 hours into imaging. I do believe the medium we had been using, Schneider’s Insect media, is optimal for the differentiation of drosophila embryonic gonads ex vivo. In the future, I plan on doing a titration experiment to find what concentration of FBS is optimal for differentiation and growth in culture media.

I was also able to track divisions over time and cell movement over time using the program, Fiji, for several of the gonads I imaged that differentiated. Below are some images analyzed using the program:

Tracking Hub Cell (t=0)
Tracking a Hub Cell (t=0)

Tracking Hub Cell (t=4.5)
Tracking a Hub Cell (t=4.5)

Figure G (t=7.5)
Germ Cell (t=7.5)

Figure G (t=8.25)
Germ Cell Division (t=8.25)

I also made some interesting discoveries regarding in vivo imaging of gonads using phytagel. I found it is possible to image developing drosophila embryos over a period of at least 8 hours using the laser scanning confocal microscope. However, I found that this dries out the phytagel, causing significant drift in the sample through the x, y, and z planes. To resolve this problem, I covered the phytagel in a coat of halocarbon oil. This did in fact significantly reduce drying out of the gel and drift. Next, I want to try zooming in more on the gonads within the embryos since drift has been reduced significantly.

Comments

  1. aealindsay96 says:

    This all sounds very exciting, Anna! Did the gonads die because of the combined M3 medium and the FBS, and if so, do you know why? Why is it that the scanning confocal microscope dries out the phytagel? Also, one more question, sorry! I’m sure you’ve probably mentioned this in an earlier blog post, but why this research on drosophila gonad development? Is there a way it this research can be applied to human stem cell development?

  2. anwesterhaus says:

    Sorry to take so long to reply! To answer your questions, it is possible that M3 media combined with the FBS caused the gonads to die. We know that the gonads cannot survive in this media as well as in media containing Schneider’s Insect Medium. These two media contain different amino acids and inorganic salts. M3 media has more amino acids and fewer inorganic salts than Schneider’s Insect Medium. M3 media may be missing an inorganic salt important for gonad development. The reduced gonad viability is unlikely caused by the FBS since the same type of FBS used with the M3 media was used in successful trials.

    The laser scanning confocal dries out the phytagel because the lasers used to scan the gel quickens the evaporation of the water in the gel. This causes the gel to shrink. Increased humidity counters this process.

    For your final question, the purpose of my research is to determine how different cells, both somatic and stem cells, move and divide within the germline stem cell niche. This work is done in drosophila since the germ-line stem cell niche is highly conserved among invertebrates and vertebrates. That means that the work done in fruit flies is relevant to humans and can help inform studies in human stem cell therapies and cancer research, and infertility research. Fruit flies have a fully sequenced genome and are easy to work with making them an ideal model system to use in stem cell niche research.

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