Prof. HORIE Takeo’s report (part 2 of 4) How to work your way up surrounded by star researchers 2022.10.12

Experiments that turn established theories on their heads

"I'm not sure if that's entirely true or not"

I created transgenic ascidians that express fluorescent proteins in Prof. SASAKURA's lab. When I was a student, I utilized antibody staining, but with that method, you can't see living samples. To stain and stabilize the structure of the ascidian tissue after killing them using formalin, a method called fixing, even took 2 to 3 days.

But in Prof. SASAKURA's lab, I inserted genes that encode fluorescent proteins into the ascidian genome, creating a lineage of ascidians with glowing nervous systems, then observed them under a microscope to study their neural circuits. The fluorescent protein used in this process, Kaede^*, changes from green to red (like the trees in autumn) when exposed to UV light, which can be used to track or trace certain cells.

• ※Kaede is a type of Japanese maple tree, famous for the variety of available green and red cultivars, as well as how some types change from a deep emerald to a fiery red in autumn

About eight hours after emerging as larvae, ascidians undergo metamorphosis: their body shape changes dramatically, and they become adults. Based on older observations, it had been said that larval brain cells were lost during this process and that a new adult nervous system was created. But I was unable to be convinced because this idea was based on looking at a series of fixed samples, so I used Kaede proteins to look at live changes in the nervous system.

After staining the central nervous system of ascidians from green to red during their larval stages, I raised them to juveniles (adults) over 3 to 4 days. If the central nervous system of the larvae was able to be preserved through the life cycle process, red-stained cells would remain, but if the central nervous system was destroyed, no red cells would remain (newly created cells would not react under UV light) and only green cells could be observed. When I checked the result of the process, I could easily see that red cells had remained. Meaning, I discovered that during metamorphization the central nervous system of the larvae is preserved and involved in the development of the adult nervous system.

From Developmental Biology to Nature

From my point of view, labeling and tracing neurons was just an observation experiment. As the paper developed from it was lengthy, about 80 pages and 20 diagrams, my first thought was to submit it to Developmental Biology^*. When I sent it to my co-author, Prof. SATOH Noriyuki of the Okinawa Institute of Science and Technology Graduate University (OIST), he told me that I had been the first to apply such an experiment to ascidians, one that couldn't be done on vertebrates, so submitting to Nature would be a better fit. I didn't like the idea; it would mean I would have to summarize the paper into 7 or 8 pages. But Prof. SASAKURA helped me cut it down. In any case, I thought that if I fail to get it published with Nature, I can try Developmental Biology after.

About 90% of papers submitted to Nature are tossed by the editor, and rejection letters are extremely quick to arrive. I've had the experience of submitting a paper at night and getting a notice by the next morning. But 1, then 2 weeks went by with no response from Nature. About a month later, I got a notice that my paper was going under peer review, and I thought that that was a good sign, at least. About a month after that notice, I got another that completely took me by surprise.

• *A core journal of developmental biology. At a time when online journals did not exist, there were no limitations on diagrams, and charges for color printing were not that expensive.

The most exciting moment of my life as a researcher

Of 3 peer reviews, I got through on my first go with the reviewer saying, "I wanted to try this experiment with mice." The second reviewer (clearly an ascidian researcher) said I hadn't completely flipped previous knowledge on its head and rejected my paper, and the third thought my paper was interesting but wasn't sure if it was right for Nature so they took a neutral stance and left the decision to the other reviewers. With a 1:1:1 result, the final decision came down to the editor, who rejected my paper.

I had given up, thinking that Developmental Biology had been the right choice after all. But Prof. SATOH told me that passing the first round was very rare, so I should appeal the decision. So, I did. The editor sent my paper in for another round of reviews, and I experimented with everything the second reviewer had mentioned. They were rough experiments, but my wife greatly helped me. A reply came about a month later.

The first, second, and third reviewers accepted my paper, though the third left the decision to publish to the other reviewers, and ultimately my paper was accepted. Being accepted within a month of checking data for a paper and paper submission is extremely fast and extremely rare for Nature.

Getting the notice of acceptance was the most exciting moment for me as a researcher. In the field of ascidians, only a few Japanese researchers, including Prof. NISHIDA of Osaka University, have been able to publish in Nature as the first author. I was deeply moved to be able to have my paper published in Nature, a publication I admire. This was my 4th year as a post-doc.

My paper was published at the time I had been looking for my next position. Soon after, by chance, I accepted the first call for open recruitment for an independent position as an assistant professor at the University of Tsukuba's Shimoda Marine Research Center.

I had started as a post-Doctoral Researcher of the Japan Society for the Promotion of Science, promoted to assistant professor, and was allowed to have my own laboratory by the age of 32. I had published in Nature and gotten my own lab in the same year, 2011. As this was only 4 years since completing my degree, I consider myself extremely lucky.

Another turning point

Research funding and a slump

When I became independent, I had 1 million yen from JSPS for research and a completely empty lab. I applied for more research funds, but, unfortunately, I wasn't selected for any. The only interview I was invited to attend was JST's PRESTO. It was for the area "Development and Function of Neural Networks" under Prof. MURAKAMI Fuji, who was part of the graduate school I was also a member of. I had been submitting applications to JST without success for the past two years. The timing of the third call for proposals and my publication in Nature coincided, and I was accepted, though the fact that I had just become independent and needed funds was probably taken into consideration. I have a great appreciation for Prof. MURAKAMI and all the others who had a hand in selecting me at that time for PRESTO to further accelerate research on the ascidian nervous system. This, I believe, was another turning point for me.

PRESTO researchers are almost exclusively mammalian neuroscientists. An outsider, like me, didn't know anyone. I was told the feeling was mutual. Still, as I got to know others, they came to acknowledge me in part as a neuroscientist and I was able to make some friends. 10 years on, I still maintain those relationships. We even get together and camp during the summer! When I was selected for my current position at OU as a professor, a member of the PRESTO team helped me practice for my interview and gave me a lot of advice on how to discuss the position. I am very grateful for my connections with PRESTO.

Then, I got into a slump.

PRESTO has a conference twice a year where all the stars of mammalian neuroscience gather. These scientists had publications in journals such as Nature, Cell, and Neuron, and here I was: a nobody with a small lab manned only by myself. I really took to heart that I had to be doing the same kind of work as all my peers. Since I had published in Nature, I figured that I needed to publish a paper in a high-impact journal, so there was a long period in which I did not publish papers when I should have; instead, I tried to accumulate them and make them into that "big paper." I was in my early thirties, the second youngest in my field selected for PRESTO, couldn't manage money well, and pressed myself to impress my peers, leading to me being unable to publish any papers. I really regretted this period of my life.

This time, I’m going abroad

While losing my way and struggling to get a paper published in the highest possible journal, I heard about an international tenure track^* position to be offered at the University of Tsukuba. As part of the program, one would have to work on the tenure track from overseas and become a permanent professor after returning to Japan. I thought this was a great position and a chance to do research overseas, so I applied, and got in.

But it was really hard. I was glad to be able to choose my own destination; however, the performance review process was more rigorous than it had been in Japan. You have to publish a good paper, so I chose a mentor with a well-known lab who had already done so: Michael Levine at UC Berkeley (current, Princeton University). He is a Drosophila (fruit fly) researcher and one of the discoverers of homeobox genes. A huge name in gene expression regulation and development research, he proposed that enhancers are very important for tissue-specific gene expression.

• *Tenured employment provides a career path for young researchers to gain experience in an independent research environment and become full-time faculty members upon final review.