Mitinori Saitou is a professor at the Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University. His research over the last two decades focused on understanding the mechanisms that regulate specification, proliferation, development, and function of germ cells in vitro. His laboratory was the first to succeed in reconstituting the mouse germ cell specification pathway in culture and to generate functional male and female gametes from embryonic stem cells and somatic cells reprogrammed to induced pluripotent stem cells (iPSC) [1, 2]. He has since expanded on these groundbreaking achievements by improving technology, expanding to species other than mice, and providing new clues regarding germ cell development. We asked him to reflect about his career progress and current challenges, future goals, and significance of research in germ cell field. When did you first become interested in germ cells and what made you want to become a scientist to study them? When I was a graduate student (1995–1999), I was working on the structure and the function of one of key cell-to-cell junctions, tight junctions (TJs), in the laboratory of Prof. Shoichiro Tsukita at Graduate School of Medicine, Kyoto University. Prof. Tsukita was a prominent cell biologist, who was most famous for his work on the identification of the integral membrane proteins constituting TJs (Occludin, Claudin family, and Tricellurin) . During his undergraduate and graduate studenthoods, he had looked at many cell types and subcellular structures throughout the body using various techniques involving electron microscopy and has acquired extensive knowledge on all the cells constituting our body. He cultivated a strong philosophy that “a beautiful structure is associated with important functions.” Based on this idea, he established a method for biochemically purifying a fraction enriched in cell-to-cell junctions from rat bile canaliculi  and succeeded in isolating many key molecules constituting cell-to-cell junctions, including integral membrane proteins of TJs . I was very much fascinated by his work and strategy, and joined his laboratory after graduating with a medical degree from Faculty of Medicine, Kyoto University. While working on TJs, I searched for an “unexplored structure” that is beautiful and may have key cellular functions, and which I could explore during my future career. When I was reading a textbook of developmental biology, I came up with a structure called “germ plasm,” which when inherited upon embryonic cleavage divisions in organisms, such as flies and frogs, appears to instruct the inherited cells to become germ cells. I found this structure and concept very interesting, and thought that if I can biochemically isolate “germ plasm” and determine its molecular components such work may uncover the mechanism on why only germ cells can eventually become “totipotent” and transmit genetic information. However, subsequently, I noted that germ cells or eggs in mammals lack the “germ plasm,” and mammalian germ cells are induced into pluripotent cells perhaps by a signaling mechanism . Furthermore, I learned the concept of genome imprinting and its erasure and addition during mammalian germline development [6, 7], which, I think, is the origin of the concept of “epigenetic reprogramming” in the germ line. I also noted that the structure often referred to as “nuage,” which is somewhat similar to the “germ plasm,” emerges in mammalian germ cells colonized in embryonic gonads . I felt that all these observations were very interesting, and importantly, very little was known regarding the mechanism responsible for mammalian germ cell development at that time. I realized that the work on “germ plasm” itself would progress through genetics using organisms such as flies and worms, and because I studied medicine and was interested in mammals, I decided to work on mammalian germ cell development, with the initial focus on the mechanism for the specification of mouse primordial germ cells (PGCs). You made many important contributions to the germ cells field. Which of them do you consider the most important and why? It is not really easy to pick up just one, because our work has been progressing in a step-by-step manner so that a new project/work has always been based on the findings of a previous project/work. But if I must pick up one, I would select the work by Ohinata et al. when we were at RIKEN CDB (2003–2009) . In this work, we clarified a principle on how germ cell fate in mice is induced in epiblast cells by signaling molecules coming from extraembryonic tissues and demonstrated that essentially all the epiblast cells at around embryonic day (E) 6.0 are competent to take on the germ cell fate in response to the key signaling molecule, bone morphogenetic protein 4 (BMP4). At this time, the precise mechanism for germ cell specification was not known, and both one-step and two-step signaling mechanisms were postulated [10, 11]. Our work unambiguously demonstrated that one signaling molecule (BMP4) in competent epiblast cells is sufficient for inducing the germ cell fate and the epiblast cells bear such competence from E5.25 to E6.25 . Remarkably, in this work, we also demonstrated that the PGC-like cells induced from the epiblast ex vivo contribute to spermatogenesis when transplanted into testes of neonatal mice lacking endogenous germ cells , serving as the foundation for subsequent strategies inducing the germ cell fate from pluripotent stem cells in vitro [1, 2]. What was the most exciting moment in your research career to date? There were many exciting moments, which are also difficult to compare to each other. I started my work on mouse PGCs with Prof. Azim Surani at the Gurdon Institute in Cambridge, with the aim of identifying key genes for PGC specification (1999–2003). At that time, there were no genes known to be expressed specifically in PGCs upon their specification. As PGCs are few in number (∼40 cells) upon their specification, I decided to take on the strategy for differential screening of single-cell cDNAs generated from PGCs and their neighboring somatic cells. I cut out a small fragment of an embryo expected to contain PGCs, dissociated it into single cells, picked up single cells randomly, and generated many single-cell cDNAs. However, it turned out to be very difficult to tell which cDNAs were generated from PGCs, as there were no specific markers. I checked expression of many markers including Oct4 (Pou5f1), Tnap (Alpl), T, and Bmp4, but their expression among many single-cell cDNAs did not give a consistent pattern leading to a potential identification of PGC cDNAs. When I discussed this result with Azim, he remembered a seminar given by Dr Kirstie Lawson, who made pioneering contributions to the origin of mouse germ cell lineage by clonal analysis . Azim said to me that Kirstie told him that some gene was specifically “repressed” in PGCs but expressed in neighboring somatic cells, but he forgot the gene name. So, after this discussion, Azim asked Kirstie about the gene and she very kindly let us know that it is Hoxb1. I immediately examined the expression of Hoxb1 in my single-cell cDNAs and found that the single-cell cDNAs are clearly divided into two types, one positive for Hoxb1 and the other negative for Hoxb1, and the Hoxb1-negative cells tend to have higher Tnap expression. I was very excited with this result and convinced that the Hoxb1-negative cDNAs are derived from PGCs . The genes specific to/highly expressed in PGCs including Ifitm3 (fragilis), Dppa3 (Stella/Pgc7), Prdm1 (Blimp1), and Prdm14 were identified from these cDNAs [12–14]. In RIKEN CDB, we improved the single-cell cDNA amplification method, with Kazuki Kurimoto and Yukihiro Yabuta, in terms of representative and quantitative amplification of original mRNAs so that it can be applied to microarray analysis . It was a stunning experience when we first looked at the heatmap of genes differentially expressed by PGCs and by their neighboring somatic cells, where all the genes that I identified by classic differential screening were included and so many more genes defining the properties of PGCs . Our method has subsequently been adapted to single-cell RNA sequence , which is so prevalent in current life sciences. We moved to Kyoto University in 2009 and initiated the work on in vitro gametogenesis with Katsuhiko Hayashi and Hiroshi Ohta. The moment when I first looked at the spermatozoa derived from PGC-like cells (PGCLCs) induced from embryonic stem cells (ESCs) in a microscopy room of my laboratory, with Katsuhiko and Hiroshi, in Spring 2010, was also so impressive that I cannot forget it, and I remember that we commented that this should be the beginning of the in vitro gametogenesis . Do you have any role models that inspired you during your career development and/or inspire you now? As I said earlier, I truly respect the science of Prof. Shoichiro Tsukita in its philosophy and quality, as well as in quantity . I also highly respect Prof. Azim Surani for his long-term passion for germ cell biology and many seminal contributions to the field. What, in your opinion, are the biggest challenges of the reproductive biology field, and germ cell field in particular, and how do you envision overcoming them? In my opinion, the biggest challenge in the reproductive biology field may be to realize the in vitro reconstitution of the entire developmental period from zygotes to term in mammals. In the germ cell field in particular, in vitro reconstitution of the entire oogenesis and spermatogenesis in as many animals (in particular mammals) as possible should be the biggest challenge. I am personally interested in the differences in the mutation rates between germ cells and various somatic lineages, and feel it will be important to uncover the mechanism for their regulations. I am also very much interested in the time differences required for the development of germ cells among different species, e.g., between mice and humans, and it will be fascinating to explore this aspect using in vitro reconstitution systems. If you were to choose a single, most important research goal that you personally would want to achieve, what would it be? My current personal research goal is an in vitro reconstitution of human oocyte and spermatogonia development. Your group was the first to develop functional male and female gametes from somatic cells using mouse as a model. How close are we to apply this strategy to other species including humans? As described in responses to the above two questions, I believe that the application of the concept and the strategy that we have shown in mice to other mammals including humans will be a key challenge in the germ cell field in coming years. It has been becoming clear that the properties of pluripotent stem cells that may be used as starting material for in vitro gametogenesis differ significantly among mammals, and the mechanism and the timeframe required for germ cell development are also quite divergent among mammals. Embryonic gonadal somatic cells that are critical/currently essential for in vitro gametogenesis are not readily available in animals other than mice. I therefore think that continuous, careful studies on various aspects of germ cell as well as gonad development in other species, including the development of relevant reproductive technologies such as spermatogonial stem cell propagation and in vitro oocyte growth, are essential to apply the mouse strategy to other species, and such efforts are in their incipient stages [18, 19]. It is, however, always possible that things progress much more rapidly than we currently assume. Gametogenesis in vitro is of great interest and importance to us scientists but manipulations of gametes or embryos often raise public concerns. Could you comment on how you see the importance of germ cell research in regard to these concerns? Very recently, we have described our ideas on the relevant issues in . Being so successful requires a lot of work hours, especially in Japan. How do you cope with it? Do you have any interests outside the lab? Do you have a recipe for a balanced life for an accomplished scientist? I have been very lucky to be able to work with many talented, highly motivated colleagues including those whose names I have given in my earlier responses. It has been a great privilege and so enjoyable to have them as colleagues. Performing experiments and discussing the results with them in the lab have therefore been and continuously are a great fun. I usually take Sundays off and spend time with my family (wife, son, and daughter). They are somewhat used to the state that I am at home only early in the morning/late at night and on Sundays, but I just enjoy very much the time I spent with them. I commute to the lab by car, and it is a ∼15 min drive. On the way back home, I often listen to my favorite music, playing it in the car very loudly. This is a moment that I can really be relaxed and like very much. What advice do you want to give to next generation of reproductive biologist/germ cell biologists to help them succeed? I think that the minimum requirements may be to study/work passionately and hard and to become confident in yourself, believing that you have the best knowledge and expertise in what you are working on and in your own field. Additionally, I think it important for you to have broader knowledge and interests in more diverse areas of biology and science in general, which will always help you to evaluate objectively the significance of your own work and to gain insight into the directions of your next investigations. References 1. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012; 338: 971– 975. Google Scholar CrossRef Search ADS PubMed 2. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011; 146: 519– 532. Google Scholar CrossRef Search ADS PubMed 3. Takeichi M. Shoichiro Tsukita: a life exploring the molecular architecture of the tight junction. J Cell Biol 2006; 172: 321– 323. Google Scholar CrossRef Search ADS PubMed 4. Tsukita S, Tsukita S. Isolation of cell-to-cell adherens junctions from rat liver. J Cell Biol 1989; 108: 31– 41. Google Scholar CrossRef Search ADS PubMed 5. Lawson KA, Hage WJ. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found Symp 1994; 182: 68– 84; discussion 84–91. Google Scholar PubMed 6. Surani MA, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984; 308: 548– 550. Google Scholar CrossRef Search ADS PubMed 7. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 1984; 37: 179– 183. Google Scholar CrossRef Search ADS PubMed 8. Eddy EM. Germ plasm and the differentiation of the germ cell line. Int Rev Cytol 1975; 43: 229– 280. Google Scholar CrossRef Search ADS PubMed 9. Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T, Saitou M. A signaling principle for the specification of the germ cell lineage in mice. Cell 2009; 137: 571– 584. Google Scholar CrossRef Search ADS PubMed 10. Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 1999; 13: 424– 436. Google Scholar CrossRef Search ADS PubMed 11. McLaren A. Signaling for germ cells. Genes Dev 1999; 13: 373– 376. Google Scholar CrossRef Search ADS PubMed 12. Saitou M, Barton SC, Surani MA. A molecular programme for the specification of germ cell fate in mice. Nature 2002; 418: 293– 300. Google Scholar CrossRef Search ADS PubMed 13. Ohinata Y, Payer B, O’Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A, Saitou M, Surani MA. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 2005; 436: 207– 213. Google Scholar CrossRef Search ADS PubMed 14. Yamaji M, Seki Y, Kurimoto K, Yabuta Y, Yuasa M, Shigeta M, Yamanaka K, Ohinata Y, Saitou M. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet 2008; 40: 1016– 1022. Google Scholar CrossRef Search ADS PubMed 15. Kurimoto K, Yabuta Y, Ohinata Y, Ono Y, Uno KD, Yamada RG, Ueda HR, Saitou M. An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res 2006; 34: e42– e42. Google Scholar CrossRef Search ADS PubMed 16. Kurimoto K, Yabuta Y, Ohinata Y, Shigeta M, Yamanaka K, Saitou M. Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev 2008; 22: 1617– 1635. Google Scholar CrossRef Search ADS PubMed 17. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Meth 2009; 6: 377– 382. Google Scholar CrossRef Search ADS 18. Saitou M, Miyauchi H. Gametogenesis from pluripotent stem cells. Cell Stem Cell 2016; 18: 721– 735. Google Scholar CrossRef Search ADS PubMed 19. Ishii T, Saitou M. Promoting in vitro gametogenesis research with a social understanding. Trends Mol Med 2017; 23: 985– 988. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org
Biology of Reproduction – Oxford University Press
Published: Feb 1, 2018
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