Farewell to Professor David Yaffe – A pillar of the myogenesis field

It is with great sadness that we have learned about the passing of Professor David Yaffe (1929-2020, Israel). Yehi Zichro Baruch - May his memory be a blessing. David was a man of family, science and nature. A native of Israel, David grew up in the historic years that preceded the birth of the State of Israel. He was a member of the group that established Kibbutz Revivim in the Negev desert, and in 1948 participated in Israel’s War of Independence. David and Ruth eventually joined Kibbutz Givat Brenner by Rehovot, permitting David to be both a kibbutz member and a life-long researcher at the Weizmann Institute of Science, where David received his PhD in 1959. David returned to the Institute after his postdoc at Stanford. Here, after several years of researching a number of tissues as models for studying the process of differentiation, David entered the myogenesis field and stayed with it to his last day. With his dedication to the field of myogenesis and his commitment to furthering the understanding of the People and the Land of Israel throughout the international scientific community, David organized the first ever myogenesis meeting that took place in Shoresh, Israel in 1975. This was followed by the 1980 myogenesis meeting at the same place and many more outstanding meetings, all of which brought together myogenesis, nature and scenery. Herein, through the preparation and publication of this current manuscript, we are meeting once again at a “David Yaffe myogenesis meeting". Some of us have been members of the Yaffe lab, some of us have known David as his national and international colleagues in the myology field. One of our contributors has also known (and communicates here) about David Yaffe’s earlier years as a kibbutznick in the Negev. Our collective reflections are a tribute to Professor David Yaffe. We are fortunate that the European Journal of Translational Myology has provided us with tremendous input and a platform for holding this 2020 distance meeting "Farwell to Professor David Yaffe - A Pillar of the Myogenesis Field".

David's pioneering studies established in vitro models to analyze rodent myogenesis 11,12,[16][17][18] and contributed to our research community the most valuable and widely available myogenic cell lines. 19,22,29,30 Along with many years of researching cellular and molecular aspects of developmental myogenesis,  David applied his lab discoveries to the cellular management of Duchenne Muscular Dystrophy 26,58,59 and neurological disorders. [67][68][69][70][71][72][73][74][75][76]88,[91][92][93][94][95][96][97][98] David's contributions have inspired, and will continue to enhance, the broader field of "regenerative myology". 100,101 David's appreciation of nature extended far and beyond lab research. His love of nature is a vivid aspect of his personality that I have carried with me since the first day I met him in the summer of 1973, when he agreed to accept me as a graduate student. Just like biological research in the lab, our surroundings offer so much, even to the naked eye. We just need to look around and discover their beauty as it is there, waitingno need to take far away trips to other continents, just look around, and see the flowers, the trees, the views, the history, and preserve it as the best as you can. Some of you may remember the myogenesis meetings David organized in Israel, starting with the first ever myogenesis meeting (1975) at Shoresh. 102 At these meetings, in addition to the amazing scientific program, David always included sightseeing tripsto see nature, archaeological sites, history, humanity. I was a member of David's lab during the years 1973-1976. I started my research by investigating the potential of Xenopus oocytes to serve as a translational machinery to analyze differentiation-associated gene expression as directed by injected mRNA / polyribosomes isolated from rat myogenic cultures. Apart from huge tub of water in which the female Xenopus frogs were swimming, I had to invent all the other tools required to make the system work. I also had to provide the nourishment to the frogs, and went now and then to the local butcher to purchase bovine liver to feed the swimmers. Using the Xenopus oocyte system, I was able to demonstrate an efficient translation of control RNA (globin), but the hope of translating the high molecular weight (200 kDa) sarcomeric myosin heavy chain was not fulfilled. I then moved to a somewhat easier system to work with, the wheat germ cell free translation system, which enabled me to discover and further study, myogenic-specific gene expression of sarcomeric myosin light chains (Z. Yablonka and D. Yaffe). 28,32 I was fortunate to have the support and input of wonderful team members. In particular acknowledgements are due to Gania Kessler-Icekson and Bruce Paterson who were already well into their doctoral and postdoctoral studies, respectively, when I joined the lab. Yaffe's group at that time was composed of a vibrant group of students, postdocs and technicians. Our research was focused on transcriptional, post-transcriptional and translational controls of myogenesis, using the most advanced methodology of that time. We worked hard with plenty of room for independent research and creativity. Some of the "ground rules" were difficult for the young spirit, but ended up serving as good lessons for years to come. Important findings were not merely dependent on picking a statistical test, but had to be very clear and obvious and reproducible numerous times. Results with cell lines were never good enough and had to be researched further with primary cells before any publication could be considered. Manuscripts often needed to be left in a drawer for a year and then be revisited to see if the report was still of interest and value.
Indeed, there were days when we came out of David's office storming and even crying, ready to never come back again, yet, the next day, the sun was shining again and we were ready to continue. I admit, without any reservations, that the years at David's lab were the best years of my scientific creativity, enabling me to focus solely on the research, with nothing else in the way. David was not just a mentor to lead students to the next level. He knew us and our families, he approved (or disapproved) of our boyfriends/girlfriends, he attended our weddings, he complimented us, and at times, made us angry, but we survived and continued and have stayed in touch with him and never thought the day would come when he would no longer be here (just as children think of their parents). For me in particular, our last names certainly have kept us together for many years. Whether it was by the Hebrew or English alphabet, we were always listed next to each other at gatherings and scientific meetings, "Yablonka" and "Yaffe" -I was delighted with this "linked" status.
I left David's lab forty five years ago, yet lost my mentor just nowon the personal level, one more touchstone of my "childhood" is gone. Here I write about him as a scientist -David was highly respected internationally and was sought after by visitors to the Weizmann Institute. As a scientist, David had original and innovative ideas that served as a basis for his research during his long career. The biological system that he developed, and which served as the basis for his work, was to culture mononucleated cells that he could induce to fuse and differentiate into muscle fibers. To accomplish this, he first isolated myoblasts from animals and learned to culture them such that they would multiply in culture. He discovered that he could change the media conditions such that the cells would fuse into muscle fibers. This system served as the basis for David's research in studying the mechanism of cell fusion and the formation of the myotubes. This was followed by establishing myogenic cells lines. One of the cell lines that David founded 43 years ago together with Ora Saxel Fuchs, is named C2. This cell line continues to serve as the basis for many research projects around the world, having served as a model system in more than 2000 publications to date. During the 70's and 80's of the previous century, David organized a number of international conferences on myogenesis in Israel. Those conferences were so successful that the participants decided to organize similar conferences in Europe.

Acknowledgments
In recent years we worked together on the gene that, when mutated, causes Duchene Muscular Dystrophy. Among other discoveries, we found that the gene is expressed not only in muscle, but also in brain. This helped explain the reduced IQ in a subset of the Duchene patients. Another important finding was our identification of a small segment of the gene that is independently expressed in the brain. Other groups found that when that portion is absent IQ is lowered further. I am sure that David will be remembered for his scientific work as well as for the C2 cell line that he established, which continues to be the basis for tens of studies a year around the world.
Yhei Zichro Baruch -May his memory be a blessing. We were two years after the Six-Day War and David was deeply concerned about the repair of massive muscle loss in the wounded. His idea was to achieve vast muscle regeneration by the provision of externally grown myoblasts, particularly the so-called 'normal' myoblast cell lines recently generated by him. We injected the cells into freeze-wounded muscles of graft-rejectionsuppressed rats and realized with great frustration that the injected cells developed into tumors. What a disappointment for David! The next step was to inject neonatal primary myoblasts grown in culture into wounded muscles of syngeneic rats. We obtained nice integration of radioactively-labeled neonatal myoblasts within the heavily injured muscles that were fully regenerated. Despite the promising results with normal myoblasts and the clear proof of concept, David's disappointment of the cell-lines was such that the project was abandoned and was never published, except in a conference lecture and a paragraph in a book chapter. I was a shy beginner and did not insist on continuing the project. For my PhD thesis, I studied the stability and translateability of polyadenylated RNA in differentiating myogenic L8 cells; part of the research was in collaboration with David's postdoc, Rob Singer. 31 When I arrived at the lab, the rat myogenic lines were already developed. Our goal was to generate a mouse myogenic cell line. Toward this goal, I prepared myogenic cultures from C3H mice. After several passages I noticed an impressive clone with amazingly looking "beautiful cells" and further isolated this clone which was named C2 -"C" comes from the donor mouse strain C3H and "2" reflects the approach (the initial cultures were established from the muscle 2 days following its injury). The article describing the development of this cell line was published in 1977 in Nature (D. Yaffe and O. Saxel). 30 Following this publication, we received inquiries from many international scientists and the C2 cells were readily sent to numerous laboratories interested in using this cell line to carry out research on the regulation of myogenesis. Subsequently, the cells were deposited at ATCC (American Type Culture Collection) and have become easily accessible and available to all. The number of published papers with the C2 cells reflects their immense contribution to scientific research advancement. Although he was already a full Professor in his 50's he liked nothing more than to look at my muscle cell cultures under a microscope, check with me how the differentiation was going, or look for some clones in my cell culture plates. He also was never afraid of new challenges. At this time David and his collaborator Professor Uri Nudel were trying to work the molecular basis of muscle cell differentiation, cloning muscle specific promoters and analyzing promoters. David who spent much of his career before the structure of DNA was even known, took up the challenge willingly, talked about sequences and cloning and RNA expression as if it was second nature. 54 One of the important papers at the time that they published looked at evolutionary conservation between Actin from chicken to human. 55 I returned to the lab in 1992 as a postdoc and David was again taking up new challenges. Together with Uri they had become leaders in the field identifying short isoforms of dystrophin, that at the time had recently been identified as the gene which when mutated causes Duchenne muscular dystrophy. David's wide ranging intellect was willing to try to test the functional role of this short isoform (named Dp71 by David and Uri) in a transgenic mice model. 72 Again what seemed like a long shot resulted in an important paper in Nature Genetics on Dp71 function. 75 But aside for a keen scientific instinct and a willingness I had the honor of being one of the last graduate students of David Yaffe. I joined his lab on January 1995 to study the role of Dp71, a shorter product of the Dystrophin gene discovered in David's lab. For that, we generated KO mice, which were the first viable KO mice in Israel. 78,83 I cherish the period of my graduate studies in which I've learned how to 'think' biology. David taught me how to ask the right questions and to observe cells at large and myogenic cells specifically. After my postdoc I returned to David's lab to start my tenure track as a staff-scientist at Weizmann Institute of Science. This was a great opportunity for me to continue working with David on our mutual interest of studying the biology of myogenic cells. During this period I've gained more knowledge understanding the biology of the muscle stem-cell population and their capacity to give rise to other cellular lineages. David was very much up to date in all cutting-edge technologies and didn't lose his endless curiosity and motivation to establish new collaborations and receiving funds to support his research. The period in David's lab has imprinted a mark on my scientific work up to this date, and opened the door for me to the research field of differentiation and dedifferentiation programs. 87,92,93 Besides science, I also acquired from David the great passion of hiking and the love of 'simple' nature, cherishing blooming of flowers and birds singing. He'd shown us some unique hidden places throughout the country in which I travel up to now, and for me they will always be "David's spots". These cells were my main tool along the years and I have never forgot to cite and knowledge David for this major contribution to the muscle field. In 1996, even though I was still kind of a "fresh woman" in the myogenesis community, David invited me to be part of the organizing committee of the Myogenesis EMBO meeting that was held in Ein Gedi. 103 For me it was a great honor but also quite a challenge, but we were all very happy that this meeting was a big success. David and I never had any mutual collaboration but from time to time we had met, he always showed interest in my research and also in my personal life. He always kept his "young" look and attitude.  [1976][1977][1978][1979][1980][1981]. It is my honor to put in writing some of my memories from those years. When I joined the lab, David was already known for establishing myogenic cell lines that could be used as a model system to study myogenesis. The myoblasts were initially cultured from newborn rats or from chick embryos. David found appropriate conditions to induce myogenesis in primary cultures on a very strict and predictable timeline, and then managed to establish rat cell lines that behaved similarly. Those cell cultures were induced to differentiate by changing media that initiated cell fusion into myotubes accompanied by the synthesis of the muscle specific proteins. Furthermore, these proteins assembled properly and produced myotubes that would even contract on the dish. The ability to repeat experiments with these cell lines led to a long and productive career during the course of which David trained many students and postdocs. I was privileged to be among them. David was a cell biologist with remarkable biological intuition beyond his direct mechanistic knowledge of cellular processes. In the search for how the myogenic developmental system worked, he focused on aspects of gene expression that were novel at the time. For example, it was clear to him that a likely control point in the expression of the contractile proteins should be the controlled production of the RNA that encodes those proteins. He thus set out to measure that RNA using the only method at the timecell free translation of RNA extracted from differentiating cultures and running the resulting (radioactively labeled) proteins on SDS polyacrylamide gels. By manipulating the cultures and by treatment with actinomycin D that prevents mRNA transcription, we were able to show that it is likely that indeed, control of myogenesis is at the transcriptional level. One day in 1977 or so, David returned from a conference abroad and was all excited. He had learned that techniques were being developed to isolate specific sequences of DNA that could be used to directly identify and measure the level of particular mRNA species in the cell. It was just the beginning of the molecular biology revolution, and David immediately understood that the new technologies that were being developed could advance our research tremendously. I remember him standing in the doorway of our lab explaining to me what he had just learned. His excitement was contagious. In short order we decided that I would drop what I had been doing since I joined the lab and that I and others in the lab would attempt to learn and perform the new technologies so that we could apply them to the questions that we were studying. Those were exciting times. David went on a sabbatical to Stanford from where he sent us protocols of techniques (like Northern blots) before they were published. We encountered many obstacles, but ultimately were able to clone cDNA's and the genes encoding alpha and beta actin, myosin light chains, myosin heavy chain, and tropomyosin. We used those cloned DNA segments to probe the RNA expression during myogenesis and published quickly. While there were competing groups abroad, we stayed ahead for the most part. David organized a conference in Israel that brought together most of the leading myogenesis research groups in the world. Our group starred at the conference, thanks to David's leadership. On a personal level, David became a friend as well as an advisor. He was an Israeli to the core. He had lived through the birth of the State and participated in its wars. He loved the country and thus organized group outings from time to time. He was a member of a kibbutz (communal farming settlement) near the Weizmann Institute and as such participated in the communal responsibilities there. This involved kitchen duty in their dining room, guard duty and the like. On the one hand he was a professor and on the other a farmer.

Authors contributions
Z.Y.-R. initiated, researched information, collected materials and contributions, and finalized the manuscript. Z.Y.-R., D.K. and D.G. translated (Hebrew to English) and/or edited some of the contributions. All coauthors discussed their contributions with the primary author during the preparation of this manuscript. All authors edited and approved the final manuscript.