CCPA Summer Series 2018: Fundación Instituto Leloir

CCPA Summer Series 2018: Fundación Instituto Leloir

Loving the Smell of Fly Food in the Morning or How To Dissect an Organ Too Small to See

By Federico Perelmuter

This summer I’ve been interning at the Fundación Instituto Leloir, in Buenos Aires, Argentina (aka home). There, in the Wappner Lab, I’ve met some wonderful people, and learned a lot about my country’s scientific system, which I ‘escaped’ in attending Haverford. The main focus of my experience was the analysis of the role of autophagy (the process by which cells eat some bits of themselves for energy and raw materials) in the differentiation of blood cells in the lymph gland. What is a lymph gland? It is a fantastic model for studying the different factors influencing the differentiation (or lack thereof) of blood cells in flies. It is an organ located at the base of the brain of Drosophila melanogaster (fruit fly) larvae, measuring a glorious 200×20 um (about as long as a human hair is wide, and as wide as ¼ of a sheet of paper is thick), and semi-transparent. I had to extract at least 6 in half an hour, in order to make experimentation worthwhile.

In my last week at the lab I carried out a full dissection protocol, from crossing the flies (basically sticking a bunch of them in a labelled jar and waiting until they produce offspring) to extracting glands from the resulting larvae, immunostaining and fixing these glands, and then mounting them into microscope slides to be photographed for fluorescence.

I thought I’d tell you exactly how you can also tear apart innocent fly larvae to observe an essentially negligible part of their anatomy, all by yourself and for no reason at all. Necessary elements for this experience are: fly larvae, 2 high-precision tongs, a microscope with up to 4x magnification, PBS buffer, and ice. I’ll leave some pictures the team and I have taken over the past months, so you can see the beautiful fruits of your labor. Needless to say, please don’t actually try this, possible side effects include accidental breakage of anything throwable around you, tears, and yelling at nearby pets.

Step 1: Picking the larvae

  • Using your high precision, ultra-sharp tongs pick out larvae that you see roaming about the edges of the bottle. You need them to be in L3 wandering development stage, the specific stage at which they begin to crawl out of their food, which is a congealed paste of polenta, agarose, water, and more, deposited at the base of the bottle or vial. This will be when they are largest, before they develop a pupa (to blossom, not into a butterfly, but a fruit fly), and also when their lymph gland is most accessible.
  • Deposit the larvae in a drop of PBS buffer on a flat surface so they remain suspended and easily visible. Repeat for as many larvae as possible.

Step 2: Tearing it apart

  • Place under microscope; illuminate from the sides!
  • Using one of your tongs, press the larva’s midriff in order to cut it in half, and then separate said halves leaving only the top.
  • Using your left-hand-tong, loosely secure the top of the portion of the larva you still have, and with the other tong, tear its cuticle off to leave a messy tangle of unknown organs floating in the PBS.

Step 3: Finding the brain

  • In the aforementioned mess, you will see a blob that seems to be composed of 3 parts, two spheres and an elongated cylinder. Try to somehow distinguish this blob from its nearly-identical-but-useless fellow blobs and isolate it.
  • Now, find the cord that emerges from beneath the two spheres. Believe it or not, the lymph gland is actually attached to that effectively invisible string, around 2 um away from the brain, and if you look closely you can see it. You probably can’t, and that’s OK.

Step 4: Separating the gland

  • Try to cut the cord at the base. Fail. Try again. Fail again. Take a small break, breathe; try again. Keep failing,
  • Once you have managed to cut the cord off, congratulations! You probably completely destroyed the gland which we wanted to analyze, but if you didn’t, you have now successfully dissected a lymph gland.
  • Dip surviving bits of nothingness into 900ul of PBS. Add 100ul formaldehyde. Cry because you may have succeeded, but probably not. Keep trying.

If one thing is clear from this ordeal, it is that persistence, patience, and care can lead to huge discoveries, in lymph gland research and beyond.

Although I came to Haverford intending to major in biology, I have realized thanks my experience at the Wappner lab that this is not where I wish to develop professionally and have decided to major in English and philosophy instead.

Tedious and enormously frustrating procedures like this one are commonplace in biology and seemed a fitting metaphor for the way I faced this internship; that is, as a trial-by-fire for my vocation, one that has allowed me, in fact forced me, to fail, and fail, and fail, until I eventually procured that ethereal, semi-invisible nugget that is growth, both personal and intellectual, from where I least expected it. I cannot thank the Wappner Lab enough for these incredible 13 weeks, as well as the CCPA for helping me through the Liberal Arts in the Workplace grant.