Beatrice Mintz, a pioneer in the fields of mouse developmental genetics and cancer biology, died on 3 January 2022, nearing her 101st birthday. I was her only graduate student. Here, I provide my memories of my time in her lab and my view of her place among the most creative scientists who have worked at the intersection of development and cancer biology.
I found my way to Bea's lab via a circuitous route. I had entered the graduate program in Human Genetics at the University of Pennsylvania and was rotating in the laboratory of Willys Silvers, known for his work on mice coat colors (Silvers, 1979). One day, while working on skin-grafting experiments with Will, I mentioned that my real interest was in developmental biology. Will responded that if I were really interested in development, I should go to his friend Beatrice Mintz's lab at the Fox Chase Cancer Center. Will knew that Bea had never taken a graduate student (she thought graduate students were a time sink and rarely productive). However, he talked with her and somehow persuaded her to interview me. By the time my interview rolled around, I had done some reading and was appropriately terrified but, right away, she asked me about a belt I was wearing that I had made from an old fragment of suede and a hammered frog I had found in a thrift shop. I am pretty sure that belt is why she decided to take a chance on me.
Bea loved to make things herself, and to design the instruments of embryology – for example, the perfect transfer pipette along with the perfect stand in which to sterilize and store it. She designed incubation chambers in French square bottles and gassing tools for embryo culture. For Bea, working it out for herself was one of the great pleasures of doing science.
Bea grew up in the Bronx. She was the youngest of four children in a family that migrated to New York from a small town that is now part of Ukraine. She skipped several grades in school and went to Hunter College in New York City, intending to study art history. Instead, she became intrigued with biology and graduated in that major, magna cum laude. From there, she studied for a year at New York University and then, because of Jewish quotas at universities on the East Coast, she moved to the University of Iowa for her graduate training. There, she joined the laboratory of the remarkable biologist Emil Witschi, where she worked on the frog Rana clamitans and the salamander Ambystoma mexicanum. Witschi was interested in the bipotential nature of both the gonad and the primordial germ cells, and how genetic and non-genetic factors influence their differentiation in multiple species. The description of his interests would actually work well as an introduction to my web page! Although Bea did work on the migration of primordial germ cells in the 1950s, she did not study sex determination or germ cell biology during the time I was in her lab. I think she must have recognized that she didn't have time for everything and somehow shifted these interests to me by subliminal means.
Bea continued her career as an instructor in the Department of Biological Sciences at the University of Chicago (1946-1960). During this period, she published a paper with Elizabeth (Tibby) Russell investigating the defects arising in three different stem cell populations in W mouse mutants (Mintz and Russell, 1957). At the time, it was thought that ‘pleiotropism’ might result from an effect of a gene in a single tissue that affected multiple tissues downstream. Bea and Tibby's research implied that the effect of the W gene on primordial germ cells was independent of the defects resulting in anemia, and those leading to white coat color. They raised the idea that the W gene might have only one function, but that function was used by several different cell types – a novel way of explaining pleiotropism. This was a golden age of mouse genetics, when the molecular tools we take for granted were not available, and when advances in understanding how genes control development were made strictly by deductive reasoning based on mutant phenotypes. Many years later, we learned that W encodes the tyrosine kinase KIT (Chabot et al., 1988).
By 1960, Bea had concluded that she preferred research to teaching. She won a Fulbright fellowship to study at the University of Paris with Louis Gallien, who was well known for his work on hormonal sex reversal and permanent phenotypic sexual inversion in the frog. Bea told me she ate oatmeal the entire year in Paris to save money to buy art on her walks along the Left Bank.
After this sabbatical, Bea accepted a research position at the Institute for Cancer Research in Philadelphia (which later became the Fox Chase Cancer Center). About this time, several important discoveries paved the way for the successful culture and manipulation of the mouse embryo. For example, in 1958 Anne McLaren and John Biggers showed that a mouse embryo could be cultured in vitro to the blastocyst stage. They developed methods to transfer the blastocyst to the uterus of a surrogate mother and established that it could develop normally (McLaren and Biggers, 1958). In 1959, Andrzej Tarkowski developed a method of transferring the embryo back into the oviduct of a host female (Tarkowski, 1959). By 1963, methods of mouse embryo culture had been improved by Ralph Brinster (Brinster, 1963). Citing these pioneers, Bea laid out her methods for mouse embryo culture in 1967, complete with her design for gassing chambers and glass pipette drying and sterilizing racks. Andrzej Tarkowski reported that he and Bea were both astonished to learn that each had simultaneously conceived the idea of producing mosaic mice by combining two embryos (Tarkowski, 1998). Tarkowski published his experimental ‘chimaeras’ in 1961 (Tarkowski, 1961), and Bea's first paper on fully viable quadriparental mice was published in 1962 (Mintz, 1962b). Bea named her mice ‘allophenics’, arguing that the name ‘chimaera’ referred to the monsters of mythology, with different parts from different animals. In contrast, she argued, her ‘allophenics’ were perfectly normal mice with an admixture of cells in all tissues. It was a bad idea to refer to them as ‘chimaeras’ at any time within Bea's hearing unless you had an hour to spare and were feeling sturdy.
Importantly, Bea developed a different technique to assemble her quadriparental embryos. Specifically, she used the protease pronase to remove the zona pellucida enzymatically (Mintz, 1962a) rather than using the tedious and inefficient mechanical method utilized by Tarkowski. Bea's approach led to a much higher rate of survival and a flood of innovative work studying the clonal basis of development and concepts of cell selection in vivo. Using carefully designed markers for each genotype, Bea investigated the effects of combining XX and XY embryos on sexual features (Mintz, 1968). She estimated the number of cells that give rise to the melanocyte population (Mintz, 1967), and to somites and the skeleton (Moore and Mintz, 1972). She demonstrated cell fusion in the case of skeletal muscle (Gearhart and Mintz, 1972), and she showed that allophenics would accept a graft from either of the parental strains (Mintz and Silvers, 1967).
Although these mosaic mice proved to be a highly valuable tool in developmental biology, Bea and others on both sides of the Atlantic had begun to think about methods to modify the genome of embryos. The field of mouse genetics had relied on spontaneous mutants and mutants derived from irradiation and other mutagenesis schemes. However, microinjection techniques for the one-cell embryo had been developed by Teh Ping Lin in 1966 (Lin, 1966), setting the stage for manipulation of the genome in the early embryo.
In 1974, Rudolph Jaenisch, who was then a post-doctoral fellow at Princeton University, was inspired by Bea's work. He and Bea decided to test the idea that viral DNA injected into the cavity of blastocysts would integrate in the genome and persist in adult cells (Jaenisch and Mintz, 1974). This experiment was the first to show that foreign DNA could integrate into the somatic cell genome, resulting in healthy genetically modified animals. Significantly, SV40 did not integrate into germ cells, so that the modified genome was not transmitted to the next generation. However, in 1976, Jaenisch provided evidence that adult males derived from embryos infected with the Moloney leukemia virus could transmit the integration to the N-2 and N-3 generations (Jaenisch, 1976). It was not until 1981 that germline transmission of a transgene was reported independently by several labs, including Costantini and Lacy (Costantini and Lacy, 1981), Wagner and colleagues (Wagner et al., 1981), and Gordon and Ruddle (Gordon and Ruddle, 1981).
Simultaneously, another approach to modifying the genome was being explored in several labs, including Bea's. Teratomas and teratocarcinomas (TCs) were of great interest to biologists because of the diverse embryonic cell types present in them. Leroy Stevens had developed a strain of mice (129/SvJ) with a high incidence of testicular teratoma (Stevens and Little, 1954) and had adapted some of them to grow as transplantable solid or ascites tumors, in which the undifferentiated cells were called embryonal carcinoma or EC cells. In a crucial experiment, Kleinsmith and Pierce showed that a single EC cell injected intraperitoneally could give rise to all tissues found in the original teratocarcinoma (Kleinsmith and Pierce, 1964). A continent away, Davor Solter and his colleagues, then working in Zagreb, systematically investigated how early mouse embryos, grafted into the testis capsule, could give rise to teratocarcinomas (Solter et al., 1970). Taken together, this body of work had profound implications. It indicated that TCs have a clonal origin, and that they could arise from cells with embryo-like stem cell properties. So, what would happen if, instead of introducing EC cells intraperitoneally, they were introduced into the early embryo; could they contribute to normal cell types in the animal?
Ralph Brinster was the first to report data in 1974 supporting this idea, but evidence for mosaicism across the mouse that resulted was limited to a few hairs in the coat (Brinster, 1974). Bea's experiments followed in 1975 (Mintz and Illmensee, 1975), characterized by her remarkable ability to design experiments with the maximum potential to yield clear answers. She designed her studies so that any resulting mice would carry strain-specific markers for immunoglobulins, adult hemoglobin, liver proteins and coat color. Experiments carried out with Karl Illmensee showed that when cycled through the embryo, TC cells could differentiate into normal tissue cells in many different organs. Other investigators in the field producing TC chimaeras found more ambiguous results: Papaioannou and Evans reported a high incidence of tumors in their TC chimaeras, and evidence of aneuploidy in TC cells, that limited their potential to contribute to the germline (Papaioannou et al., 1978, 1975). Despite these issues, and the skepticism about some of Illmensee's results, Bea's ideas and outstanding experimental designs really advanced the field.
In addition to the significance of her ideas to the field of embryology, Bea's conclusions had very important implications for the cancer field. They showed that a cell derived from a tumor and placed in the environment of the embryo could be ‘gentled’ and restored to normalcy. In her own words, they provided ‘an unequivocal example in animals of a non-mutational basis for transformation to malignancy and of reversal to normalcy’ (Illmensee and Mintz, 1976). Bea and others in the field (Pierce and Cox, 1978) put forward the idea that cancers arise from dysregulation or subversion of normal developmental pathways in stem cells. She suggested that in constantly renewing tissues such as the blood, the testis, the gastrointestinal epithelium and the skin, the niche microenvironment is crucial to control the proliferation versus normal or abnormal differentiation decisions in stem cells. She suggested that changes in this microenvironment might indirectly influence the behavior of stem cells, leading to cancer (Mintz and Fleischman, 1981).
In 1977, she diagramed the idea that TCs, which could be cryopreserved and propagated in vitro, could be genetically manipulated and reintroduced into the embryo (Mintz, 1977). Later that year, she tested this idea by chemically mutagenizing TCs, selecting for HPRT deficiency, then showing that those cells carrying a genetic modification could be reintroduced into the embryo and could contribute to the full array of somatic tissues (Dewey et al., 1977). However, contribution to the germline and transmission to subsequent generations remained a problem until the development of embryonic stem cells in 1981 simultaneously by Martin Evans's lab in Cambridge University (Evans and Kaufman, 1981) and Gail Martin's lab at the University of California at San Francisco (Martin, 1981).
I arrived in Bea's lab in 1982, when her primary focus was shifting to hematopoietic stem cells (HSCs). My goal was to test the idea that single HSCs, marked by a unique retroviral insertion, could give rise to the entire range of hematopoietic cell types. Instead of using irradiated hosts, we used mutant hosts with defects at the base of the hematopoietic tree [W/Wv, Wf/Wf (a Kit allelic series of mutants)], or specific to the lymphoid lineage (SCID mutants) (Capel et al., 1989, 1990; Capel and Mintz, 1989). We tested the idea that the level of engraftment would depend on available space in the niche, and a competitive advantage of donor cells. We injected bone marrow or fetal liver cells into the orbital vein of neonates, taking advantage of the period of neonatal tolerance to test histocompatibility limits between donor and graft. Bea had an uncanny ability to be standing just behind me every time I dropped my forceps, whereupon I would have the benefit of a lecture on the proper use of valuable scientific equipment, and I would be advised (again) that I should never expect a replacement of any of my surgical tools during my time in her lab.
I remember being so proud when I left my first paper on her desk. Having pursued creative writing as an undergraduate, I thought that I had at least the writing aspect of my training under control. But the following Monday Bea called me into her office and rapidly dispelled that idea. ‘Who do you think you are? Edgar Allan Poe?’, she said. ‘You have hidden all the clues in the closet. You have written a mystery novel and not a scientific paper’. To say that I learned a lot about writing from Bea does not begin to cover it, but, even today, I catch myself writing mystery stories and hearing her words in my ear.
Bea had an enormous talent for thinking about important questions in the context of her vast store of information about biology. It was always a pleasure to return to the lab after institute seminars, when Bea was excited by the talk and wanted to think more about it. She would put the talk into a completely different context for me. For example, she would remember some obscure fact about Paramecium or a lizard species that shed light on the problem at hand. At the time, I thought I should aim to accumulate that much information over a long career. Now, I see that it was not just the information, but the retrievable storage system that was so impressive.
Bea was exacting – of herself and everyone around her – and some days the best idea was to spend as much time as possible out of reach in the mouse colony. She had a strong sense of what was right and wrong and there was not a lot of gray in her life. Her Washington Square apartment was almost entirely black and white: white sofa, white carpet, ebony sculptures, and Picasso drawings from her days in Paris. On the other hand, she was often very warm – at least toward me. She had a great sense of humor, as demonstrated in her 1964 letter about ‘Yeti’ to her colleague Dr Timothy Talbot, who was the first president of the Fox Chase Cancer Center.
Bea taught me that research life is short, and that I should never waste it on experiments I already know the answer to. She had a poor opinion of embarking on experiments based on hypotheses. She felt this biased the investigator. Instead, she believed in listening carefully to your experiments. She always focused on the big questions, and she designed elegant ways to probe those questions and extract information that the average scientist would not even know was there for the taking.
She received many honors during her career (including being elected to the National Academy of Sciences as well as the American Academy of Arts and Sciences, receiving the first Ernst Jung Gold Medal for Medicine, the National Medal of Honor for Basic Research by the American Cancer Society, the Szent-Györgyi Prize for Progress in Cancer Research, and a Lifetime Achievement Award from the American Association for Cancer Research). However, she was most proud of being a recipient of the first March of Dimes prize in Developmental Biology (shared with Ralph Brinster) and of having been chosen for the Pontifical Academy of Science, where she advised Pope John Paul II about human embryonic stem cells. A picture of the Pope shaking hands with Bea hung among the tapestries in her office. I think she found this appointment most amusing for a Jewish girl from the Bronx.