Skip to content

When you choose to publish with PLOS, your research makes an impact. Make your work accessible to all, without restrictions, and accelerate scientific discovery with options like preprints and published peer review that make your work more Open.

PLOS BLOGS PLOS Biologue

Behind the paper: Courage is the best magic

In this ‘behind the paper’ post, Yuting Lei discusses how he overcome several technical difficulties in his search for the adult fat body precursors in Drosophila.

My name is Lei Yuting and I am a fifth-year PhD student from Jose’s Lab at Tsinghua University in Beijing. The new paper from our lab mainly focuses on adult adipogenesis in Drosophila, which is regulated by FGF signaling. In simple terms, our work is trying to answer two questions: 1, where does adult fat body come from? 2, What influences adult fat body precursors’ migration? For me, this project is composed of small obstacles, occasional surprises and of course, long-time but necessary exploration. Still, all the waiting is worthwhile the moment you know you found exactly what you had been looking for.  

Back to the time when I was a freshman in our lab, I knew little about adult fat body. Previous studies from our lab are mostly larval fat body-related, and we use it as a convenient model to investigate cell secretion. One “irrelevant” question that was raised several times by the audience of our previous subjects was — “we already know a lot about larval fat body, so what about adult fat body?” — I still remember that it was in April 2019 when Jose talked to me about this project, then I searched relevant papers discussing adult fat body and the last paper I found showing some experimental evidence of the possible origin of adult fat body was published in 1995 by Hoshizaki’s group. It could have been a good joke from Jose, given that April Fool’s Day had been just half a month ago. For me, it takes lots of courage to face a puzzle unsolved for decades. Things could not be forgotten in a corner for no reason. However, as a rookie my greatest strength was fearlessness and as a Chinese saying goes, a newborn calf is not afraid of a tiger. Therefore, my first reaction to this project was “Okay, why not give it a try?”

The first thing was to see what adult fat body looks like, and with all accessible tools in our hand, what is the earliest timepoint to which we could trace adult fat body. We checked previous Gal4 lines that are reported to label fat body (mainly larval fat body), but we did not find useful ones that could obviously indicate location of adult fat body precursors during early migration processes. So, we had to start from scratch. We used a lineage-tracing method combined with tub-Gal80ts to successfully label those precursors from the beginning. It is a system that could label cell lineage under control of temperature. This labeling is permanent and heritable across generations of cell division, but early activation of the system would lead to labeling of both larval and adult fat body. To avoid signals in larval fat body,  we added another “switch” tub-Gal80ts (temperature-sensitive) to turn on the system at later developmental stage through shift of temperature (18°C off→30°C on). Still, this technological path was not perfect, as we could also see signals from other tissues, especially muscles.

And here comes another practical problem — how to do long-time live-imaging of pupae when flies have opaque pupal case to protect themselves during metamorphosis. In other papers I checked, people usually partially open the pupal case in other situations. But I had to remove it thoroughly to show the whole abdomen of flies. What is the key factor to keep them alive for dozens of hours? It was already winter in Beijing, which means it was dry and cold at night. What’s worse, the stereo microscope room, where experiment was conducted, has no heating system (later, we used confocal microscope in a room with constant temperature). I got an electrical heater and an air humidifier to try to keep those naked soft pupae alive. Every night, I set up a new live-imaging device (coverslip-sample-slide in a sandwich structure held by clay), watched it over for several hours, prayed that it would make it until the next day, and returned to my dorm. And usually, the next day I would get a newly made pupal “mummy” under the microscope. Indeed, it was frustrating but informative enough to let me know that humidity is the most important factor. It occurred to me that I could use a glass-bottomed plate containing paper tissue soaked with water as a humidity chamber to protect the naked pupae. This time, the poor pupae could survive in the chamber until the end of pupal stage, which means my little handcraft did work. That’s how I got over our first obstacle for living imaging.

After removal of pupal case, the samples were put in a glass-bottom dish
Image credit

Lei Yuting, CC BY 4.0

Later, we got a glimpse of how adult fat body precursors migrate in the early stage of the abdomen. But using the lineage tracing method, we still got signals from the muscles, which is unavoidable. Also, it means we cannot conveniently do any genetic manipulation in adult fat body cells because the specificity of our technical method is not good enough. Then, the screening for better Gal4 line had to be continued. Because of the COVID-19 pandemic, it had been two years since this project began when I came across our Mr. Clutch, OK6-Gal4. In a word, we could clearly label adult fat body precursors using this cell line. We saw exactly the same population of cells we found in the lineage tracing method without any redundant signals from other tissues. The most exciting moment was the night when I got our first 10 hours’ living imaging movie recording the spreading of adult fat body precursors in the abdomen. The beauty of nature unfolds before you when those little elaborated biological machines swim orderly in the cavity of the abdomen, whose migration path looks like butterfly wings.

Adult fat body precursors migrate in the pupal abdomen
Image credit

Lei Yuting, CC BY 4.0

Without a stop, we recorded the process of adult fat body formation and started the screenings of genes that could influence the formation of mature adult fat body. We found that FGF signaling plays an important role regulating direction and substrate adherence of migrating precursors, guiding and coordinating the flow of these cells. Two intriguing directions could be derived from our recent findings: 1. Specific manipulation of adult fat body is now realizable to check adult fat body’s physiological function in adult flies. My collaborator Yuwei and I have done some preliminary work about this. 2. We provide a new model to study cell migration in vivo, which still have many genes waiting to be discovered.

There are two things that supported me to the very end of the project: one is enthusiasm; the other is patience. I still remember that during the first months, Jose and I together dissected many samples continuously, searching for clues of adult fat body precursors, and sometimes even missed lunch time. At that time, we were hungry, both mentally and physically. We have tested many ways to show those cells, and finally overcome those obstacles and found some fascinating new stuff, which is impossible without patience. Yuwei helped me a lot in studying maturation process of adult fat body and during the revisions of the paper, and we have tried different experiments together to refine our model. It is really nice to have someone supporting you in the toughest time during the COVID-19 pandemic.

About the author

Error: No image and/or text provided. Please provide content for both fields.

Related Posts
Back to top