The people behind the papers – Izumi Oda-Ishii and Yutaka Satou

Izumi (L) and Yutaka (R)

Yutaka, can you give us your scientific biography and the questions your lab is trying to answer?

YS: Following my PhD in developmental biology at Kyoto University under the supervision of Nori Satoh, I had the chance to be involved in an ascidian genome project. This experience changed the direction of my research, because I realized that I could do something new with its simple and compact genome. So I started a project to understand the gene regulatory system of this animal by combining the knowledge and skills I had acquired during my PhD and genome project. A 1 year stay in Mike Levine's lab at UC Berkeley also reinforced this direction of my research. I have been working on this problem for 20 years.

Izumi – how did you come to work with Yutaka and what drives your research today?

IO-I: Ever since I started my research using ascidians with an interest in evolution of cis-regulation of key developmental genes, I had been impressed with Yutaka's work, which explores the molecular basis of ascidian embryogenesis employing genome-wide approaches. I had the good fortune to join his lab 11 years ago, and I always enjoyed my science since then. What drives my research is the beauty of developing embryos under the microscope, which is much more than skin deep and raises so many profound questions.

How has your research been affected by the COVID-19 pandemic?

YS & IO-I: Ascidians become mature adults in 6 to 12 weeks, and we raise them in our lab and at a marine station belonging to our university both for our own use and for distribution to other ascidian labs in Japan. Our lab was closed for almost 1 month last year, and during the lab closure we minimized the number of ascidians we maintained. Several months were necessary to recover the usual population of animals. Today, regular lab meetings are still held online, and doing experiments remains somewhat restricted; however, we hope to return to normal life in the near future.

Before your paper, what was known about the binding sites and developmental roles of ascidian Zic-r.a?

YS: Zic-r.a, also known as Macho-1, was first identified by Hiroki Nishida of Osaka University as a maternal muscle determinant. In 2004, we determined the nucleotide sequences that preferentially bind ascidian Zic-r.a using an in vitro selection assay. While this maternal transcription factor activates a target, Tbx6-r.b, immediately after zygotic genome activation, definite Zic-r.a-binding sites were not found in the regulatory region of Tbx6-r.b. This has been a long-standing research problem in my lab.

Can you give us the key results of the paper in a paragraph?

IO-I: It may widely be believed that a transcription factor recognizes only a single motif. However, we show that an ascidian Zic transcription factor, Zic-r.a, recognizes two motifs to induce gene expression in different cell lineages at different developmental stages. Since distinct sets of zinc-finger domains are required to recognize the two motifs, they are not simply variants of a single motif, but distinctly different.

Why do you think neural genes, controlled by canonical Zic-r.a sites in later embryos, are not activated in the early embryo?

YS: That is one of the problems we want to tackle. Because Tbx6-r.b reporters in which non-canonical sites are replaced with canonical sites are expressed in early embryos, it is highly likely that canonical sites in neural genes have the potential to bind Zic-r.a. Epigenetic regulation may be involved, but this remains to be tested.

How common are transcription factors that bind multiple distinct sites via different domains?

IO-I: Although several studies have suggested that a considerable number of transcription factors recognize multiple motifs, this is still controversial. Zic-r.a is a transcription factor with five zinc-finger domains. Our data suggest that the modular structure of Zic-r.a is important in recognizing two distinct motifs. We do not have a clear answer to your question, but we think that transcription factors that possess such a modular structure potentially recognize multiple motifs.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

IO-I: The moment I succeeded in identifying Zic-r.a target genes in neural cells and found Zic canonical motif sites in their upstream regions, I thought, ‘I knew it!’ It was very satisfying.

And what about the flipside: any moments of frustration or despair?

IO-I: To knockdown zygotically expressed Zic-r.a without affecting maternal Zic-r.a mRNA, I first injected a morpholino oligonucleotide against Zic-r.a into anterior-animal blastomeres of eight-cell-stage embryos, in which maternal Zic-r.a mRNA is not localized. I was frustrated with this experiment because it was extremely laborious and it was difficult to obtain reproducible results with a limited number of embryos. I eventually realized that I could more easily obtain such embryos using TALEN technology.

What next for you after this paper?

IO-I: I'm interested in how transcription factors that recognize similar DNA sequences in vitro bind to different regions in vivo. The ascidian genome contains another Zic factor in addition to Zic-r.a. Although they recognize similar DNA sequences in vitro, they are believed to regulate distinct sets of target genes. I want to explore the mechanisms by which these two factors activate different sets of genes.

Where will this story take the lab?

YS: We have previously identified several transcription factors with zinc-finger domains that are important for specification of cell fate. I want to know if these factors also recognize multiple motifs with different affinities and to compare their properties in order to understand whether there are common mechanisms among them.

Finally, let's move outside the lab – what do you like to do in your spare time in Kyoto?

IO-I: I like biking along the Kamogawa river with my kids. We enjoy lunch, catching small fish, and crossing the river on turtle-shaped stepping stones. In the spring, we can admire beautiful cherry blossoms there.

YS: The Kyoto Basin is surrounded on three sides by mountains, and I enjoy walking in the woods near my house. My kids used to come with me, but they have become teenagers and rarely come so these days I enjoy walking with my wife.