Prof. HORIE Takeo’s report (part 1 of 4) You don’t have to be part of 'high society' to make it as a researcher 2022.10.5

Me, my mentor, and the sea squirt

A life full of failure

I’m a little different than other people. I think most other professors at FBS succeeded at something big when they were younger and became professors in a very short time—they were stars from the beginning.

But not me. And I want the students here to know why. There’s a part of me that hopes to be a role model for those who didn’t go to the top universities, didn’t enter a famous laboratory, weren’t research stars, and weren’t set to become a professor from the get-go—a role model for those who put their nose to the grindstone as a researcher.

I didn’t ride the elite highway to success. Not even from the beginning. The first university I entered was a public university deep in the countryside. When I had to go in for admission procedures, I took a bus from the nearest train station and fell asleep. When I finally awoke, I was in a bit of a panic that I had missed my stop, but the bus driver told me that we were still deep in the mountains. Going to a university so far out made me wonder if I could really continue there.

It wasn’t my first choice. I actually wanted to go here, Osaka University, but I had failed to get in… this is where my life of failure first started. I was all set to take a year off after high school, but a friend said to me, “I’m taking a year off because I failed to get into any university. Do you think you, who has been accepted into a university, are allowed such a luxury? It’s unforgivable.” I had already started an application to attend a college preparatory school, but his words made me change my mind at the last minute. So, I went to university. I suppose that in the end, it was a good decision.

Meeting my mentor and the sea squirt

I encountered a sea squirt on my second day of university during orientation. The man who would become my mentor and the professor in charge of my graduating class, Prof. TSUDA Motoyuki, introduced us to the sea pineapple, a type of squirt, during a tour of the campus facilities. When the teacher asked us if we knew what kind of animal he was using in his research, none of the 40 or 50 of my classmates could answer. I loved illustrated reference books, so I knew what it was. I answered, “Teacher, that is an ascidian. A sea pineapple.” The professor was overjoyed. That was the first time I met my mentor and the sea squirt. But at that time, I didn’t think I would research ascidians.

I actually met my wife at that same orientation. When I tell high school students that they might find their lifelong research project and partner one year from now, they find it pretty funny.

I wasn’t a model student in high school—I had thrown myself completely into kendo. I had hoped to get into a university on a kendo recommendation, but when I failed to get that, I turned to study. My university days were as normal as anyone else’s. I often tell my students to not goof off in school, but my wife says I’ve got some nerve putting myself on a high horse considering how I used to act in university. I wasn’t a serious student, but I had a lot of fun.

If research is this fun, I’ll be a researcher!

I'm too clumsy to research embryology.

Soon after, I visited a lab, but studying ascidians wasn’t my first choice. What I thought was interesting at that time was embryology and the nervous system. In embryology, there was only one laboratory working on planarian regeneration, and Prof. AGATA Kiyokazu (now director of the National Institute for Basic Biology) was an assistant professor. Prof. AGATA’s classes were pretty interesting, so I thought embryology was for me, but I made a huge mistake during lab work involving chicken embryos. We had to use a cutter with a blade costing 2,000 yen to cut open the chicken eggs, transfer fertilized ones into larger shells, wrap them and observe them. Since the blades were so expensive, the teacher warned us not to drop them—but no sooner had he said that than I dropped mine and completely bent the blade.

The professor got really mad. He said, "Horie! In the 10 years I’ve been conducting these practice experiments, you’re the first person to ever drop a blade! You’re clumsy. Out of all the people here, you had better not come to my laboratory."

I suppose it was a joke, but at the time I thought it meant that because I was clumsy, I couldn’t do embryology research. Prof. AGATA was soon transferred to Okayama University, so I couldn’t have chosen planarian research anyway. Then I remembered that I had always been interested in the brain and my encounter with ascidians, and I decided to work with Prof. TSUDA, who had been researching ascidian brains. At that time, Prof. AGATA was very kind to me as an advisor for a Fusion Oriented Research for Disruptive Science and Technology project. Although he was not my direct research mentor, he had a great influence on my choice of brain science and developmental biology, and I still respect him very much as I did then.

You probably understand by now that I haven’t ever been able to act on my first choice in anything in my life. You might even think I might have developed a complex about not being able to do the things I wanted to do or join the lab I wanted to, but not only did I not develop a complex, I also never lost my motivation. I think there are students who can’t get motivated unless they get into their first choice of labs, but you don't have to be discouraged if things don’t work out. I would like to tell them to do their best at whatever they choose as it leads to the next choice. And that is a positive thing. All of my choices were negative. But they lead to where I am now, a result that is positive.

Treasured pictures

Of course, I thought ascidians, sea squirts, were interesting living things. But I had no idea about their brains when I began my research. So, I came up with the idea of color-coding ascidian brains to classify neurons and study the function and development of each. I shared this strategy with my professor, but unfortunately, he did not approve it, partly because it was different from the first theme I was given. So, without telling my professor, I secretly conducted experiments in the middle of the night with the assistant professor. That's how the assistant professor and I developed antibodies to a protein in the eye called arrestin.

This is my most treasured picture. I was looking through a microscope alone in the lab in the middle of the night and saw this. Being the first person in the world to see this… me, a fourth-year undergraduate student, was incredible. I was so enormously moved. I knew then that I felt I wanted to continue my education and pursue a Master’s. Even though I had a vague idea of myself as a science-y type who should continue just because and an admiration for my peers already in graduate school, I had my doubts about getting a PhD and didn’t consider it at the time.

But in September of my first year as a master’s student, my doubts were put to rest by when I gave a poster presentation focusing on the ascidian nervous system. I used the picture taken earlier of ascidian photoreceptor cells, which were still unknown at the time, and presented my findings at a small conference. It was a great success, and many people came to see my poster. My work was also praised by Prof. NISHIDA Hiroki from the Tokyo Institute of Technology (currently, he is a professor at the Graduate School of Science, Osaka University). I couldn’t believe I was praised by such a distinguished person! It made me realize how fun research can be, and I decided to pursue a PhD and become a researcher.

At the time, the magazine "Cellular Technology" had a page titled "One Photo Gallery," and I was excited to think I would be able to use the photo of photoreceptor cells for it.

The confidence to succeed anywhere

Do whatever it takes

I still hold the same idea for research that I had written on my first application for research funds when I was a first-year Master’s student: neural circuit mapping of ascidian larvae.　 But at that time, we did not have the technology to manipulate and experiment with neural circuits at the single-cell level. That technology was developed over three years from 2015 to 2018. With a technique called single-cell transcriptome, we classified neurons one by one and were able to introduce foreign genes into single cells. I was always thinking about what could be researched at the single-cell level for 10 or 20 years, even before this technology became available. I think we’re where we are now because we jumped on the tech bandwagon as soon as it rolled in.

Prof. TSUDA taught me to do whatever it takes to achieve my goals. To accomplish things, we should not stick to past experiences and methods, but fearlessly introduce different methods and new techniques, and what I have considered since becoming a PI is similar to his methods, that is, thinking about how to introduce new technology to bring about breakthroughs. I don’t think you can create something new if you’re stuck using old technology, so I’m not afraid to bring new tech into my lab.

To get back to the topic, I had trouble in my doctoral program due to many reasons and it took me four years to finish. I imagine many students are worried that if they don’t finish their PhD by their 3rd year, they would have difficulty becoming a researcher, but anyone can bounce back. Even though it took me 4 years to finish, I don’t think a single year was wasted. I had lots of time to think, and there weren’t many distractions, so I could concentrate on my research. After obtaining my PhD, I went to the Shimoda Marine Research Center, University of Tsukuba, which is also kind of out in the boondocks. However, there is no place more rural than where I studied as an undergraduate; being there gave me the confidence to be able to work anywhere!

Three choices

After finishing my doctoral course, I had 3 huge choices to make regarding my field of study: 1, research on the development of the vertebrate eye (my first choice), 2, research pineal glands in fish, such as the zebrafish, and 3, continue studying ascidians. I discussed the possibilities of each with my academic advisor at that time. I learned that the professor studying zebrafish was an acquaintance of Prof. TSUDA’s and asked him to introduce us.

I asked the professor who was working on pineal glands for permission to conduct research on a new mechanism of developmental differentiation by comparing the retina and the pineal gland (similar but different tissues). The professor allowed it, on the condition that I was accepted into the JSPS PD Program, but I failed spectacularly at being selected for that.

I was then introduced to a researcher who was part of the discovery of Noggin (NOG), William C. Smith (Bill) from UC Santa Barbara, a world-famous embryologist and originally a researcher interested in the African clawed frog. He had moved his interest from the African clawed frog to creating mutant ascidians that lacked pigment cells in the brain and asked us to analyze the structure of that mutant brain. As I had handled antibodies that stained nerve cells and experience doing joint research, he allowed me into his lab. So, I prepped myself for a move to UC Santa Barbara.

At the same time, Prof. SASAKURA Yasunori of Tsukuba University, who was conducting similar research on mutant creation in Japan, invited me to visit his lab. The professor was inserting transposons, or moving genes, into the ascidian genome to create glowing ascidians, then moving the transposon causing the destruction of the ascidian gene. In a sense, he was in competition with Bill. I was invited to his lab, but I kept refusing, as I already had a contract with Bill's lab and would go to the United States.

To the Shimoda Marine Research Center

But then I had a lot of problems with the professor who introduced me to Bill… so in the end, I refused to go to UCSB and decided to join Prof. SASAKURA’s lab instead. Prof. SASAKURA quickly sent me employment documents before I could change my mind, and with that, I headed off to the University of Tsukuba's Shimoda Marine Research Center. I had never been to Shimoda before, and I had made a spur-of-the-moment decision. This choice, as well, was the right one in the end.

ACKNOWLEDGEMENTS

Prof. SASAKURA is a pioneer in ascidian genetics who created the method of using transposons to create next-generation ascidians with foreign genes. He was a tenure-track faculty member and established his lab when he was about 31 or 32 years old. With only one PI and one postdoctoral fellow, it was exciting to work in his lab. We were very close in age, and he trained us in everything from the basics of genetics to the fundamentals of ascidian research. Prof. SASAKURA taught my wife and I to do experiments, and we are here today thanks to him. I am still in contact with Bill—we are doing joint research together. Since last year, Bill, Patrick Lemaire, a French ascidian researcher, and I have been working on an international joint research project, "Computational Neuroscience in the basal chordate Ciona," with support from the National Institute of Information and Communications Technology.