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2 Develop. Growth Differ. (2008) 50, S3 S9 doi: /j X x Interview Interview with Dr Tokindo S. Okada in commemoration of the 50th volume of Development, Growth and Differentiation Tokindo S. Okada, Yumi Momiki, Elizabeth Nakajima and Kiyokazu Agata* Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto , Japan Even if they paid authors only 1000, DGD would instantly become famous! 1. Introduction 1.1 Atmosphere of the interview Dr Tokindo S. Okada, Honorary Adviser of the JT Biohistory Research Hall in Takatsuki City, Osaka prefecture, Professor Emeritus of the Department of Biophysics, Kyoto University, and 2007 recipient of the Japanese Order of Culture (Bunka-Kunsho), came to the North Campus of Kyoto University on a beautiful, sunny afternoon in mid-september to sit down with Kiyokazu Agata, Development, Growth and Differentiation (DGD) Associate Editor and Professor of Biophysics, Kyoto University, and a few other lucky listeners to give a special interview to commemorate the 50th volume of DGD. Dr Okada, dapper and cheery, brought to the interview a bright spirit of scientific enquiry and clear appreciation of how developmental biology has itself developed in the past 60 years or so. Two hours of listening to his accounts came to an end all too quickly, and the listeners said goodbye to him wishing to hear more of what he thinks about experiments and research pathways that led to important advances, some pathways that didn t lead to the advances people imagined they would, and the people who walked those paths seeking to understand the living creatures that presented themselves to their wondering senses and minds. *Author to whom all correspondence should be addressed. agata@mdb.biophys.kyoto-u.ac.jp Received 6 December 2007; accepted 18 December Journal compilation 2008 Japanese Society of Developmental Biologists Dr Okada had clearly presented an astute eyewitness view of how the Japanese Society of Developmental Biologists (JSDB) came to be established and how the journal DGD came to be published. 1.2 Brief Introduction of Dr Tokindo S. Okada Tokindo S. Okada (Fig. 1) was born in Itami City, Hyogo prefecture, Japan, in He graduated from Konan High School in Kobe City in Hyogo prefecture, and then became a student in the Faculty of Science, Kyoto University. He developed a strong interest in embryology in high school, when he learned biology from Dr Hirosi Takaya, who had become a teacher of biology in Konan High School after getting a DSc degree in the Department of Zoology at Kyoto University. While Dr Okada was a student in the Department of Zoology, he carefully analyzed the fate map of endoderm by traditional transplantation experiments. After going abroad to the University of Edinburgh and the Carnegie Institute of Washington, he brought cell culture and immunocytochemical techniques back to Japan, where he then pioneered cellular approaches to developmental biology. Dr Masatoshi Takeichi was strongly influenced by Dr Okada, and succeeded in identifying key cell adhesion molecules he named cadherins in Dr Okada s laboratory in the Biophysics Department of Kyoto University. In 1978, Dr Okada started molecular biological approaches to developmental biology, and since then he and his colleagues have contributed greatly to advances of molecular biological approaches in the field of developmental biology. Dr Okada was appointed the Director of the National Institute for Basic Biology in 1984, and is currently an Honorary Adviser of the JT Biohistory Research Hall. He was awarded the Order of Culture in 2007.

3 S4 T. S. Okada et al. Fig. 1. Professor Tokindo S. Okada, in June 2007, at the Department of Biophysics, Kyoto University. 2. Establishment of Japanese Society of Developmental Biologists We would like to hear about the early days of the JSDB. I know that the JSDB was established separately from the Japanese Society of Zoologists in Can you tell us how experimental biology came to be a major stream of science in Japan? How did this lead to the establishment of the JSDB? And what are the roots of the JSDB s publication, the journal DGD? Well, experimental biology gradually became a major stream of science in Japan after two great scientists who had studied abroad returned to Japan in the 1930s. They came home bringing new trends in biology from western countries. First, I ll talk about Dr Kaname Okada, with whom I share the same last name by chance. K. Okada had a big hand in the organization of the JSDB. I think K. Okada stayed in Paris in the 1920s (he went around Europe far and wide, so I can t be certain!) and did experiments using various living things in a number of famous laboratories in Europe. After coming back to Japan, he became a professor at Kyoto Imperial University. He became a sort of Kaiser, who had great authority in embryology. There is an anecdote about him, saying that he had experience using all living things except for human beings and bacteria. His rich experience enabled his co-workers in Japan to get a view of the frontiers of world science and to educate young researchers. At Kyoto University, he established a new section (a koza in the Japanese academic system) for awarding the DSc degree in experimental embryology, which started a new trend in biological studies in Japan. A lot of students joined his new section and fell under the spell of the new biology. At that time, there were a lot of wild newts, whose eggs and embryos are suitable for microsurgery, living in and around Kyoto, and, partly because of this, K. Okada s new field of experimental embryology made more progress in Kyoto than in Tokyo. One thing that slowed progress for a while and took a lot of time and effort to figure out was that the protocols that K. Okada had used in Europe employing Holtfreter s saline for the culture of amphibian embryos had to be adjusted in Kyoto, because the water in Germany, where Dr Johannes Holtfreter devised the formula for his saline solution, contained much more calcium than water in Kyoto. K. Okada thought that an academic society was needed in Japan for this new biology. Around that time, his interest moved on from embryos to adults (and incidentally, he left Kyoto Imperial University, and became a professor at Tokyo Imperial University). K. Okada had become a Kaiser, a great boss of embryology, in Japan, and he had enormous influence, so the name of the new society that was organized, the Japan Society for Experimental Morphology, reflected his interests. He loved this name very much. The society also published a scientific journal in Japanese, a good journal. Its name, translated into English, was Annual Journal of Experimental Morphology. This journal was one of the two basic roots of DGD. The other great founder of experimental biology in Japan was Dr Tadao Sato. In the 1950s, he was a professor at Nagoya University (formerly Nagoya Imperial University). He had been born into a rich family and had many good connections, and using those connections, he and the Embryologia Society published a journal of embryology in English; this was a challenging job in post-war Japan. The name of the journal was Embryologia, and it was a local letter of embryology in Nagoya University at its initial stage. T. Sato put great effort into this journal. It was the other basic root of DGD. In 1968, these two scientific societies were united and named the Japanese Society of Developmental Biologists. By that time, K. Okada had retired, so he didn t have much influence on the new society, and its name didn t include the term experimental morphology.

4 Interview with Dr Tokindo S. Okada S5 Fig. 2. Covers of first issues of the journals Annual Journal of Experimental Morphology (translation of its Japanese title) (left) and Embryologia (right), and of Development, Growth and Differentiation (center), the journal that their unification gave birth to in It took a while longer for the two journals to be unified. After the JSDB was established, the two journals, one in Japanese and the other in English, each continued to be published for a while, but within a year or so they were united and newly named Development, Growth and Differentiation (Fig. 2). Dr Masao Sugiyama was a developmental biologist who was a professor at Nagoya University and later became a professor of biology and president of Sugiyama Jogakuin University. He was a representative at the first meeting of the JSDB. He put great effort into publishing the first issue of this combined journal and he chose the cover design. As a result of publishing DGD, JSDB became more prominent as an organization, and DGD also became a publication of this society rather than of a university, a book-publishing business, or a person. Thus DGD came into the world; this name was decided as a result of a vote. Embryologia was also one of the names proposed, but it was not selected. Instead, yellow, the color of the front cover of Embryologia, which was a landmark of post-war embryology in Japan established by T. Sato, was used for the cover of the new journal. That s why the main colors of the cover of DGD are still always yellow and black. 3. Organizer fever, depression, and resolution Young researchers these days do not know the atmosphere of the 1960s and 1970s. Probably, I am in the last generation knowing about both the organizer s fever and depression. I remember very well your comment when I bought the book entitled Organizer. You said, Organizer research fascinated many young scientists and killed them! How did people manage to reactivate the developmental biology community from the ant hell of organizer research? To answer your question simply, we have to make a shrine and worship newts there! K. Okada was the one who started organizer research in Japan. He came to realize that newts were useful for many kinds of biological experiments. Experiments being essential for experimental morphology, he promoted the use of newts for studies of embryology. In those days, there were few researchers who had used newts, and no good experimental methods for using them. The researchers and students in K. Okada s lab had enormous difficulty developing nice methods for newt experiments with the aid of K. Okada s records and his memory of the experiments he did in Europe. K. Okada, the great boss of the field of embryology (experimental morphology) in Japan, focused on newts, so we can t describe experimental morphology without newts. Historically speaking, K. Okada was always seeking not the basis of individuality, but rather that of generality. He wanted to find the principles of development, not diversity. Newts played an important role in this

5 S6 T. S. Okada et al. stream of embryology in Japan. At that time, all researchers using newts were regarded as experimental morphologists, and what they really wanted to know was how different parts of an embryo interact with each other. The headstream of their studies was organizer research. K. Okada published some key papers, of which the most important one demonstrated that inserting various inorganic materials into an embryo caused the formation of something like a secondary embryo (Okada 1938). It was said that this structure was consistently something like, though not exactly, a secondary embryo, but when I saw photographs of it, I thought it was undoubtedly a secondary embryo. For one thing, it had a neural tube. Of all his papers, this paper attracted the greatest attention of researchers internationally. Another person, Dr Tuneo Yamada, also published interesting research about the organizer. He hypothesized the existence of a substance which would work like an organizer and induce a complete second embryo. The research of a Chinese scientist, Dr H. H. Chuang, who had worked in Japan, seemed to confirm this hypothesis. He put a fragment of kidney or liver in an embryo and saw that the formation of a second embryo was induced. He published an 80-page paper about this in He had gone back to China before the start of the Second World War, and he became the head of a scientific academy in Shanghai and continued to publish good papers. He was often invited to international meetings of biologists after the war. It was quite remarkable to invite someone from a socialist state to a free nation in the Cold War years, but everyone wanted to listen to his talks and to learn from him about the historical background of biological research. This paper of K. Okada s was very highly regarded among a lot of papers about the organizer, but at the same time, it decreased the ardor of biologists for discovering the actual nature of the organizer. And it s no wonder it did so. The identification of the protein normally working as the organizer in embryos would not be evaluated very highly, these scientists thought, since K. Okada s paper had shown that even inorganic substances could function like the organizer. However, some biologists maintained their desire to identify the organizer. In fact, it was no longer a mere desire: they made desperate efforts to discover it. There was one of them in Japan, Dr Makoto Asashima. He had belonged to the lab in Germany of Dr H. Tiedemann, who also had a great passion for the organizer. Different biologists used different sources to get substances which had functions similar to those of an organizer. Tiedemann and Asashima used chick embryo extract. The active substance might be described as a mysterious all-around elixir. Anyway, the elixir certainly induced some kind of organization in tissue culture, and particularly good cultures sometimes developed complete embryos. Culturing various tissues was no problem if one added chick embryo extract to the culture medium. A lot of biologists made a frantic search for the true identity of the elixir. Seeking it was a long, hard struggle. In retrospect, it s natural that embryo extract could induce various kinds of organization, because it contained all the components of a body. Biologists lost their passion for finding the organizer by degrees. A substance with so many different effects should not be a special protein, they thought. Research about a substance that can do everything would be like the expeditions sent out by an ancient emperor of China to get the elixir of life. Even so, some biologists, including M. Asashima, continued searching for the organizer until the end of organizer research. In the end, M. Asashima found activin, which functions specifically as an organizer factor alone (Asashima et al. 1990), but, more importantly, he showed by experiment that the cells of an extremely early embryo can become any part of the body, I think. He cultured parts of embryos at an initial stage of embryogenesis together with an organizer-like substance, and thereby discovered the totipotency of different parts of the embryo. Needless to say, the discovery of activin is one of the greatest landmarks of developmental biology. However, I think becoming aware of the totipotency of the embryo was also a giant step for biology, whether Asashima had succeeded in finding the organizer itself or not. 4. The path to cellular developmental biology To escape from organizer hell, many people tried various new approaches to developmental biology. You promoted cellular approaches to developmental biology. How did you decide to use cellular approaches? Did cell culture have an impact on you? When I was working in the United Kingdom, it was not worth seeking the actual substance acting as the organizer. Rather, I looked for parts of newts which

6 Interview with Dr Tokindo S. Okada S7 Fig. 3. Title page of 1939 paper of Johannes Holtfreter in the journal Archiv fur experimentelle Zellforschung that strongly influenced the research of T. S. Okada by introducing the concept of affinity between different tissues into his thinking. had not been examined yet, and I found that these consisted only of endoderm tissues. They exist in an inner location, so it s difficult to perform surgery on them. In addition, they grow at a late stage of development, so there is a need to maintain the developing newts for a long time. For these reasons, no biologist had studied endoderm in a fruitful way. Starting my research on the endoderm of newts, I took the paper of Holtfreter titled Gewebeaffinitat, ein Mittel der embryonalen Formbildung (Holtfreter 1939) (Fig. 3) as very important. Even now I think this paper is very significant. Holtfreter brought the notion of affinity between different tissues into our thinking about the processes of development. This led me to try to culture endoderm tissues and skin tissues together, and to study how it was that the two tissues in such cultures always repelled each other. Holtfreter s paper gave me the concept that such a phenomenon was important for clarifying the mechanisms of development. I came to think that cells as well as tissues can have affinity for one another. In embryological research, it was thought that surgery was very important, but tissue culture was not. However, there were some biologists who did a great deal to build the foundation for the use of tissue cultures in developmental biology research, and to make it possible to use chick embryos for it. Dr Honor Fell in England cultured a chick primordium on agar and made a limb which had an elbow and even five fingers. She showed that one could get a great deal of information about 3-D morphogenesis using tissue cultures. That was the birth of developmental biology research in vitro. Dr Conrad Hal Waddington was struck by Fell s findings and advanced the method through his research. As a result, it became possible to use chick embryos for experimental embryology. Dr Aron Moscona, who had belonged to Fell s lab, wrote the paper that most impressed me of all the research in those days. Using chick embryos, he made the very interesting finding that dissociated neural retina and mesonephros cells reaggregated with the same kind of cells, respectively (Moscona & Moscona 1952). The emphasis on observation at the cellular level was important for embryology, I thought. In his experiments, trypsin was used for dissociating cells. I tried to reproduce his research after returning home, but I had great difficulty doing it. When I told him about my big travail at a later date, he laughed and said that the trypsin he had used at Cambridge University was all important: the reagent called trypsin actually contained a lot of other materials, and one had to use a preparation containing a good blend fitting one s individual purpose. The cell biology of embryology got its start in those days, and I also helped it get started. I showed that endoderm tissues covered with skin always lost contact with each other, but that they maintained their adhesion when I added mesoblast. I also succeeded in marking cells with some isotopes before mixing two tissues. This was useful for observing the differences of the behavior of cells from different kinds of tissues. This cell-marking method received high acclaim in the UK. My stay in the UK ended at this point. When I came back to Japan, many, many embryologists were still chasing the organizer using newts. No one used chick embryos. Additionally, cell-level embryology has a kind of paradox: morphogenesis happens not in each cell, but at the tissue level. That is why other embryologists used to think I was a peculiar fellow. Anyway, I continued my research and tried to develop new methods: labeling with fluorescent antibody, for example. Through many trials and errors, cell biology studies of development advanced, and we became able to do a good job of following the behavior of cells.

7 S8 T. S. Okada et al. Fig. 4. Left to right: Yumi Momiki, Elizabeth Nakajima, Kiyokazu Agata, and Tokindo S. Okada in September 2007, at the interview on which this article is based. 5. Molecular biology and developmental genetics You also incorporated molecular biological approaches to developmental biology at a very early time. When I entered your laboratory as a DSc student in 1979, my research started with the purification of restriction enzymes. You strongly contributed to the modernization of the JSDB. Many talented researchers graduated from your laboratory. What can you tell us about why you began to use molecular biological approaches to developmental biology, and what breakthroughs those approaches led to? In the 1970s, gene recombination was born. It enabled us to manipulate genes similarly to how we could manipulate organs. I thought this technique would yield new findings in developmental biology, and I strove to make my lab be the first embryology lab to use it. Even highly competent biologists from around the world looked at my big rush with surprise. At that time, people were making a lot of noise about molecular biology: some people criticized the technique as an awful method that would create a monster. But, at the same time, it started the dawn of the new developmental biology. After this, some students who came to my program of developmental biology first began by using restriction enzymes, although they had not yet seen an embryo. That kind of thing would have been unthinkable in former times. Time passed, and gene recombination was becoming a routine method for biologists. I took up a post as the director of the Okazaki National Research Institutes. When I left Kyoto, I said to M. Takeichi, my successor there, Next, you have to find the gene for adhesion, as my last words as an active scientist. Takeichi told biologists in America about my last words, and they were surprised at what a reckless punch these words seemed to be, leaving him with a sort of black eye. But, after a time, Akira Nagafuchi, a fellow worker in my Kyoto days, came to Okazaki and said, We got it! I asked him, What is it? That s how I got the news that they had discovered the gene for cadherin, a key protein working in cell cell adhesion (Nagafuchi et al. 1987). My last words had become reality. I was very surprised. What fantastic news! I thought my old-time associates who had made great efforts searching for the cadherin gene were amazingly efficient scientists. Now, cell sorting and gene recombination have become quite ordinary methods. But in the stream of great advancements of biological techniques, in my life as a scientist, I was somehow always at the front of the movements. 6. Concluding remarks Thank you for giving us (Fig. 4) this unique account of the genesis and advances in cellular biological studies of animal development. This is a special interview for the 50th anniversary of DGD. So, I would like to ask whether you have any comments regarding the anniversary. Near the end of my research life, I published a paper (Kondoh et al. 1983) in a journal published by Springer-Verlag and received a fee for it. In Germany in those days, academic and artistic publication was a

8 Interview with Dr Tokindo S. Okada S9 protected cultural field. The publishers thinking was that academic papers, like musical scores, would retain monetary value and academic significance indefinitely. So they paid authors. That s not going to happen nowadays. Maybe I was the last Japanese who received a fee for publishing a paper in those good old days. Nowadays, authors are required to pay an excess page charge when they publish their scientific papers. Suppose DGD would start to pay authors some fee for original papers! Even if it were just 1000, I m sure that DGD would instantly become famous. I think my research life could be rather well described by the Japanese idiom Ki-Sho-Ten-Ketsu, which is a special type of idiom consisting of four words, namely, in this case, Introduction (Ki), Development (Sho), Turning point (Ten), and Conclusion (Ketsu). That is, I started my research life with the paper of Holtfreter demonstrating the phenomenon of cell cell affinity, worked in the field of cell biology, employed new techniques, including gene recombination, and ended with the memorable event of my former colleagues finding the gene for a key protein mediating cell cell affinity. That discovery by Takeichi and colleagues has always seemed to me clearly to have been the Ketsu. My research life has been pretty closely synchronized with the 50 years of DGD. I participated in the combining of the two journals and the birth of DGD, and now am joining this event of the 50th anniversary, although I have already stopped being an actively working scientist. I m deeply thankful to have been a part of it all. Conflict of Interest No conflict of interest has been declared by T. S. Okada, Y. Momiki, E. Nakajima or K. Agata. References Asashima, M., Nakano, H., Shimada, K. et al Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor). Roux s Arch. Dev. Biol. 198, Holtfreter, J Gewebeaffinitat, ein Mittel der embryonalen Formbildung. Arch. Exp. Zellforsch. 23, Kondoh, H., Takagi, S., Nomura, K. & Okada, T. S Transdifferentiation of putative neuronal cells of neural retina into lens: a demonstration by chick-quail chimeric cultures. Roux s Arch. Dev. Biol. 192, Moscona, A. & Moscona, H The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J. Anat. 86, Nagafuchi, A., Shirayoshi, Y., Okazaki, K., Yasuda, K. & Takeichi, M Transformation of cell adhesion properties by exogenously introduced E-cadherin cdna. Nature 329, Okada, Y. K Neural induction by inorganic matters, with special reference to the mechanism of induction through the introduction of a foreign object into the blastocoele. Mem. Coll. Sci. Kyoto Imp. Univ. Series B 14,