Prof. HORIE Takeo’s report (part 3 of 4) The most I’ve ever researched in my life – my study abroad 2022.10.19

The most advanced place in the world

Living the dream at UC Berkeley

Michael "Mike" Levine was working on gene expression regulation in Drosophila (fruit flies), but he also used ascidians to study evolution.

Before entering Mike’s lab, I took a tour of UC Berkeley. I thought California was a great place, and there happened to be two other Japanese in Mike’s lab. One of them was about my age, Dr. YAMAZAKI Yuji, who had left his position as an associate professor at the Graduate School of Medicine at Osaka University to study at Mike’s lab.

I spent days wandering the campus and dreaming of life under the blue California sky with my family. However, In December of the year before I was to depart in April for my actual study abroad, Dr. YAMAZAKI sent me a mail asking if I was surprised to hear that Mike was moving to Princeton University. I had no idea! Nor did I know where Princeton was. Google told me it was on the East Coast. Oh, well. So much for my dream.

But once I learned what a nice place New York was, I told Mike that I would be following him to Princeton. I started getting my Visa documents ready in a hurry, but I didn’t have a good idea of where Princeton was, and I had never been, so preparing everything was difficult. At this time, it just so happened that Mike was in Japan as a lecturer for a winter course at the Okinawa Institute of Science and Technology Graduate University (OIST), so I took the opportunity to talk to him there. He told me that Princeton is an affluent university in the US, with a budget about the same size as Harvard's, but twice the allocation, since Princeton has half the number of students and faculty. Then he said, "Takeo, you can buy whatever you need. The money flows freely." That last part turned out to be a joke. Haha. We also talked about what I was to do at Princeton. When I set on UC Berkeley, I was going to study neural circuits, but my destination at Princeton was the Lewis-Sigler Institute for Integrative Genomics. He told me that he was thinking of doing something with single-cell transcriptome^* at Princeton, something he couldn’t do at UC Berkeley. This was near the end of 2014.

• *Single-cell transcriptome (analysis): A technique to comprehensively examine gene expression in each individual cell, allowing the classification of neurons one by one and the introduction of foreign genes into a single cell.

I could do all the research I had wanted to

All at once, I was able to do the research I wanted to. I went to the US and started working on single-cell transcriptome analysis. It was a small lab with only 15 to 20 people, but everyone came from other famous laboratories. My Princeton experience was truly irreplaceable. I belonged to the Lewis-Sigler Institute for Integrative Genomics and the Department of Molecular Biology where weekly seminars were held to which famous researchers from around the world were invited to attend. I realized I was in an environment with global, cutting-edge research when I saw the incredibly fast pace that unpublished data was being presented in those seminars.

A particularly memorable event was when I attended a closed research meeting on transcription and toured the Janelia Research Campus, operated by the Howard Hughes Medical Institute. Also, Eric Betzig, a group leader at the Janelia Research Campus, was awarded the Nobel Prize in Chemistry for his super-resolution microscope while I was there. I had so many valuable experiences, such as being shown the Lattice light-sheet microscope^*, which was not commercially available at the time. Unreleased data presented in closed research meetings caused heated discussions to develop, and that data was then published in Cell, Nature, Molecular Cell, and other publications one after another within half a year.

I thought, "This is research in America." It was a feeling I got to experience firsthand. Working in one of the world’s top labs and experiencing such exciting things every day, my wife and I often forgot to eat and sleep and threw ourselves completely into research.

But since this was America, I also took care of my family, attended all of my children's daycare open schools, took breaks when it was break time, worked when it was time to work, and devoted myself to research more than I ever had, or have since, in my life.

• *Light-sheet microscopy (like confocal microscopy) allows three-dimensional imaging of samples and is used to analyze the structure of the cranial nervous system. It is characterized by its ability to capture images quickly with minimal damage and from multiple angles, though with lower resolution than confocal microscopy. Lattice light-sheet microscopy maintains the features of light-sheet microscopy but provides a higher resolution.

Introducing new techniques one after the other

Successfully creating artificial dopamine neurons in ascidians

It is said that there are two organs, the neural crest cell and the head placode, which are the most important in vertebrate evolution, and the acquisition of those two organs is the most important for the evolution of vertebrate species. Eyes, noses, mouths, teeth, jaws—our faces—are characteristic of vertebrates. The cranial placode creates eyes and noses and such, while the neural crest cell creates teeth and the cranium. At what stage of evolution these organs were acquired, or which has an earlier origin, was not well understood. To try and unlock this puzzle, I began research on ascidians.

Looking at ascidians’ evolutionary tree, ascidians are invertebrates most closely related to vertebrates and retain ancestral traits, thus can exhibit the properties of common ancestry of both. Based on that logic, we studied whether ascidians have neural crest cells and/or cranial placodes and, if so, what ancestral characteristics they exhibit. We published our findings in Nature in 2018.

In 2018, we successfully produced artificial ascidian dopamine neurons. We used single-cell transcriptome techniques. I think the paper was very well received and contributed in a major way to where I am now.

I have so much gratitude for Mike for the many things he taught me. I think I may never experience such an exciting and stimulating time again. Submitting my paper to Nature was the most memorable part of my time with Mike. We worked on it together, and when it was time to submit, he said, "Takeo, take my name off this paper. If you leave it, people will think that you were able to get published because of me. This is your paper." I explained that the aim of the International Tenure Track Project was to have participants develop a paper alongside their overseas mentors and that we needed to submit this paper together. Although I ended up submitting the paper with his name on it as well, just the idea of him volunteering to remove his name makes him a magnificent person.

"Discovery depends on technology"

A journal called Development interviewed Mike^* for an article about showcasing newly discovered technology and put him in the spotlight. Mike's lab, when I was there, was using single-cell transcriptomes for ascidian research and live imaging of transcription for Drosophila (fruit fly) research. Transcription studies had always been examined using fixed samples, but now, live imaging is a method that allows the live observation of when and where transcription occurs. I am currently applying this live imaging technique^** to ascidian transcription. Studying single-cell transcriptomes with Mike led us to start a new research project on transcription. Now, that project’s research was selected for the FOReST (Fusion-Oriented Research for disruptive Science and Technology) program, and a new research theme has developed.

• *"An interview with Mike Levine"
• **『セントラルドグマの新常識』（羊土社）(transl. "Central Dogma’s New ‘Common Sense,’ " Yodosha, Inc.)

Though this narrative so far may feel like a success story, it was not smooth sailing. I came to the US in June to change my address from Berkeley to Princeton, but it still took a month for my things to arrive. Also, nobody understood anything I was saying at first. Even though I was told I could buy anything I wanted, that wasn’t quite true, so I had to do a lot of work to get the things I needed for my experiments. Setting up a lab in the US was much more difficult than in Japan. But due to this experience, I felt that setting up my lab (in Japan) was so much easier.

"accept in principle" means another round of peer reviews!?

Mike was discovered to have a heart condition while I was putting together my tenure track paper, so he had to leave the lab and work remotely from his home in Berkeley for a period of time. It was exhausting. I had to revise my paper four times. After working on getting my paper published for a year, when the editor finally said, "We are delighted to accept in principle your paper," I felt as if a long battle had finally finished rather than being happy about it. And the feeling of relief was stronger, too, as I sensed that my tenure track position would now be set.

But I got a letter from my editor the following week saying that after having an editor’s meeting, they decided that since there weren’t any single-cell transcriptome experts assigned to my paper during peer review, they would need to do another round of peer reviews that included a transcriptome expert. I was a bit shocked. Mike became furious, as he hadn’t ever heard of Nature doing something like this.

So, then I had to wait another nerve-wracking month.

The paper was accepted, but I was exhausted by the tenure track review process, and I thought that I didn't want to do tenure track projects again. Despite the ups and downs of my time in the US, I was able to return home with tenure.

Forced transfer

A cramped room

I was happy to return to Japan, but it was difficult to work because of how small the lab I was given was. It was 15 square meters. After setting up my office and my wife's worktable, there wasn’t even space for us to pass each other. The left half of another room was also my space. It was so cramped, we couldn’t pass each other there, either. Even with this work environment, I managed to submit a paper to the Proceedings of the National Academy of Sciences (PNAS). Still, working in such a space was draining. Even if we wanted to increase the number of lab members in the future or hire a post-doctoral fellow with the large research grant we received when we came to this position, there was no space for them. I had three students, but there was no space for their experiments, so they borrowed the lab of a professor also researching ascidians. There was no way to gather more people under those conditions, so I made the decision to transfer.

Another huge reason for my decision was that the tenure position I received was for an assistant professor. So, I searched for associate professor positions in the surrounding universities, but I couldn’t get hired anywhere. When I consulted with an acquaintance, they encouraged me to try for a professor position instead. So, for the first time, I took the plunge and applied for a position as a professor at the Graduate School of Frontier Biosciences, Osaka University. After receiving advice from the members of PRESTO, I went for an interview and was hired. Everyone tells me that my journey is very encouraging. Now they know it is possible to go from an assistant professor in a small lab at a regional seaside experimental station to a professor at a large lab like the one I am working in now.