HOLTFRETER, JOHANNES

(b. Richtenberg, Germany, 9 January 1901; d. Rochester, New York, 13 November 1992),

experimental embryology, inducers and organizers, tissue affinity.

Holtfreter was one of the best experimental embryologists of the twentieth century. As a student of Hans Spe-mann, Holtfreter was trained in a German tradition of experimental embryology. His research defined and addressed issues of fundamental importance in embryology, such as how different embryonic tissues are induced to change, how cells coordinate movement during development, and how tissues recognize each other. With the rise of national socialism in Germany, Holtfreter immigrated to the United States where he continued to develop a reductionist approach to embryology.

Early Years . As the only son in an upper-middle-class family in northeastern Germany, where his father owned a whiskey factory, Johannes Friedrich Karl Holtfreter spent his childhood exploring the countryside not far from the Baltic Sea. His interests in nature developed into an interest in biology in the Realgymnasium and then the University of Rostock and the University of Leipzig, where he studied until 1919, when he transferred to the University of Freiburg. At Freiburg, Holtfreter studied and worked for Hans Spemann, who was quickly becoming established as one of the world’s leading embryologists.

In 1924 Holtfreter completed an uninspired thesis on organ development in frog embryos. At the same time, Hilde Mangold performed the foundational experiments on the organizer that would win Spemann the Nobel Prize in 1935.

Spemann was interested in when and how cell fates and structures are determined in the early embryo. Spe-mann and his group focused on embryos in the blastula stage, when they form a hollow ball of cells, but before gastrulation, when cells move inward through the blasto-pore to create different germ layers and the basis for the body axis and nervous system. Mangold used Spemann’s technique of transplanting groups of cells from one part of the blastula to another in order to determine the developmental potential of those groups of cells. Most of these transplantations had slight effects. When she transplanted cells from the dorsal blastopore lip to the opposite side of the blastopore, however, she discovered that a second body axis began to develop at the transplantation site. Moreover, Mangold and Spemann determined that the transplanted cells had not only continued to develop themselves but had induced the surrounding tissues to change and form part of the new structures. Spemann called this area above the blastopore the organizer, because of its ability to influence or induce changes in surrounding tissue. Spemann and Mangold published their results in 1924 just as Holtfreter was finishing his own graduate research.

Holtfreter was not immediately drawn into organizer research. Spemann, who did not approve of Holtfreter’s frequent escapes to hike in the forest, suggested that Holtfreter go to the Naples Zoological Station to study marine biology and embryology there. Using his own funds, Holtfreter traveled to Italy and ended up on the island of Ischia in the Bay of Naples, where he avoided doing any science and instead painted frescoes in the village church in St. Angelo. Holtfreter was a very talented artist, and after two years in Italy he returned to Germany and actually tried to support himself as a portrait painter for awhile.

Job prospects in biology were not good for Holtfreter, especially without Spemann’s full support. Holtfreter considered working in a fisheries research station in Helgoland and as a high school teacher. However, in 1928, when he despaired of finding any future in science, Otto Mangold, Spemann’s favorite student and Hilde Mangold’s spouse, invited Holtfreter to join him as a researcher at the Kaiser Wilhelm Institute for Biology in Berlin-Dahlem. Holtfreter immediately accepted and began one of the most productive periods of his research life soon after.

Kaiser Wilhelm Institute . Spemann’s organizer experiments had set off an explosion of research in experimental embryology, and Holtfreter entered the conflagration as a member of Mangold’s group. Where Spemann’s approach was guided by an idea of the organism as a functional whole, Holtfreter was interested in the developmental potential of the different regions of the gastrula in isolation. Dissecting thousands of embryos into parts and following the development of each part in isolation met with an immediate technical difficulty. Embryos had been cultured in just filtered water and tended to die quickly. Holtfreter needed his embryo sections to grow for a longer period of time in order to determine if any differentiation occurred, so he conducted a series of trials to create a sterile salt solution that allowed cultured embryos to develop much more fully. This solution, which has since become known as “Holtfreter solution,” made it possible to carry out much more significant embryological research using amphibians.

One of the key questions raised in organizer research concerned what properties enabled the organizer to induce changes in surrounding tissues. Spemann was sympathetic to the idea that some aspect of the organizer’s structure was responsible and in 1931 reported the results of some transplantation experiments using crushed organizers. Holtfreter extended this line of thought with dramatic effect. He killed organizers with heat, cold, and desiccation and then transplanted them into blastula in his solution. The killed organizers induced new neural structures and decisively demonstrated that the inducer was not a property of living tissue, and therefore must be a chemical. In addition, Holtfreter undertook transplantation experiments with adult tissues and tissues from other species and phyla. Many of these were capable of neural induction as well. This very productive period of research in the 1930s raised important new questions about the organizer as it shifted research away from the properties of the organizer as a group of cells to the search for inducing substances. In effect, Holtfreter’s induction experiments created biochemical embryology.

The impact of Holtfreter’s research was widely appreciated, and in 1934 he was appointed to an associate professorship in the Zoology Department at the University of Munich. After only a year in Munich, Holtfreter took advantage of a Rockefeller Foundation fellowship and a private travel grant to spend a year at Yale University as a member of Ross Harrison’s laboratory. Harrison and Spemann were very close friends, and Harrison had been a frequent visitor to Freiburg. Even though Holtfreter knew Harrison and his research program, he was not deeply interested in the kind of tissue culture research that then dominated Harrison’s group at Yale. Instead, after a few months, Holtfreter took his leave to explore the United States. Drawn westward, Holtfreter traveled by train to California and then by steamer to Hawaii and Bali. Captivated by the island and its people, Holtfreter stayed in Bali for several months. Only when his funds ran out did he continue his journey back to Germany.

War Years . The Germany Holtfreter had left was not the same to which he returned. The Nazi Party had continued to consolidate its power in the mid-1930s. To Holtfreter, going from Bali to Nazi Germany was like moving from paradise to hell. Although he was not Jewish or overtly political, he was soon subject to harassment from the Gestapo. In 1938 he presented his research at the Congress of Physics, Chemistry, and Biology at the International Exposition in Paris. As the most notable German participant, he was informed that he was Germany’s representative to the congress. Holtfreter nevertheless presented his paper in French and then took the honorarium and explored Algeria before returning to Munich, actions that put him in danger. Once he was back in Germany, he was denounced and imprisoned. An international effort secured his release and emigration in 1939 to England, where he joined Joseph Needham’s group at Cambridge University. As the war escalated, however, Holtfreter was classified as an enemy alien and placed in an interment camp in Canada, where he remained until 1942.

Holtfreter later described the two years he spent in a Canadian prison camp as the worst years of his life. Unable to do research and too demoralized to read the scientific literature that was sent to him, he painted landscapes of Bali on wooden shingles. In 1944, with the support of John Berrill, a biologist at McGill University in Montreal, Holtfreter was released from internment and hired at McGill. Old friends such as Ross Harrison rallied around Holtfreter, shipping him amphibian eggs and embryos to restart his research program. After adjusting to his new circumstances, Holtfreter turned to the topic of gastrulation.

Research on Gastrulation . In the 1920s Walter Vogt had used stained living embryos to follow the patterns of cell movement, involution, and germ layer formation. This complex process of gastrulation appeared orderly as the hundreds of cells harmoniously coordinated their movements. Vogt and later Spemann suggested that gastrulation was not merely wandering cells but the result of a superior force that directed the motions of the cells. Holtfreter was skeptical of this approach to gastrulation, just as he had been skeptical of Spemann’s account of the organizer. At McGill he applied his techniques of dissection and tissue culture developed in the 1930s to cells involved in gastrulation. As he studied clumps of cells and the movements of individual cells, Holtfreter devised what he thought of as a mechanical explanation for cell movement in gastrulation. Noting that bottle cells tended to elongate, Holtfreter proposed that the invagination of cells at the blastopore was initiated by bottle cell elongation. Other cells on the blastula surface followed the bottle cells into the interior as the result of a coating that bound surface cells together and so produced the apparent coordination of their movement.

Like his earlier work on induction, Holtfreter’s research on gastrulation pushed experimental embryology to embrace a more reductionist and analytic understanding of the processes of development. Holtfreter’s postwar papers on gastrulation clearly expressed his dissatisfaction with Spemann’s and Vogt’s view of gastrulation as planned and coordinated by some supercellular agency. In its place, Holtfreter explicitly chose to frame gastrulation as a mechanistic phenomena with cellular and physico-chemical causes.

In the mid-1940s Holtfreter extended his analysis to processes of neural development in early embryos. He wished to understand whether the organizer region was really essential for neuralization. Lester Barth had demonstrated in 1941 that ectoderm could be induced to develop into neural tissue by simply changing the salt concentration of the surrounding solution. Using his earlier technique of culturing isolated amphibian embryonic tissues, Holtfreter subjected ectodermal fragments that would normally form nervous tissues to different concentrations of salt with additional changes in the pH of the solution. By systematically adjusting the chemical conditions, Holtfreter was able to induce ectoderm to form not only neural tissue but sense organs.

Because these salt solutions were presumed to be free of any chemical inducers from the organizer, these experiments demonstrated that ectoderm possessed a capacity to form neural tissues independent of the action of organizer tissues. Holtfreter proposed that the salt solutions he used destroyed the ability of ectoderm to suppress their capacity to form neural tissues, resulting in what he called autoneuralization. The observed ability of this tissue to form recognizable neural tissues and structures convinced him that the ectoderm itself was an important source of organization and structure. These experiments on autoneuralization furthered the shift among experimental embryologists away from the organizer as the active determiner of embryonic structures to the inherent capacities of responding tissues.

University of Rochester . In 1946 Curt Stern, a fellow émigré from Germany and an old friend from the Kaiser Wilhelm Institute for Biology in Berlin-Dahlem, persuaded Holtfreter to leave McGill and join the faculty at the University of Rochester in New York State. Having not been particularly happy in Canada, Holtfreter looked forward to joining other émigrés in the United States. He spent the rest of his life in Rochester, eventually marrying Hiroko Ban, a biologist and former graduate student.

In Rochester, Holtfreter turned again to a series of experiments he had done in Germany in the 1930s. In 1939 Holtfreter had discovered from his long series of isolation experiments that like cells from different structures and germ layers had definite affinities. Mesodermal tissues, for instance, were attracted to both ectodermal and endodermal tissue, while ectoderm and endoderm seemed to avoid each other. This cellular affinity was strongest among older cells that had undergone more differentiation.

At McGill Holtfreter had noted that the cells from isolated embryonic tissues could be separated into an assemblage of free cells. Given a chance under normal conditions, these cells rearranged themselves into tissues very close in structure to those that had existed originally. In the 1950s Holtfreter and his student Philip Townes returned to this research of cellular affinity and the selective adhesion of cells with each other. Using isolated cells, Townes and Holtfreter mixed cells from different germ layers and observed the patterns of reaggregation. They noted both the affinity and movement of different cell types as they sorted themselves into distinct tissue layers. Ectodermal and endodermal cells again demonstrated no affinity for each other but strong affinity for like cells. Mesodermal cells, by contrast, adhered to both endoderm and ectoderm and so resulted in a germ layer arrangement typical of developing embryos with mesoderm sandwiched between the endoderm and ectoderm.

By following the movements of cell types, Townes and Holtfreter demonstrated both preferential movement and association. As with his other embryological work, Holtfreter concluded from these experiments that the organization of germ layers was the result of cellular processes, not higher-level capacities or features of the organism as a whole. Townes and Holtfreter’s paper profoundly influenced research on embryonic morphogenesis by refocusing attention away from germ layers to cells and cell types that make up those germ layers. As with his earlier work on induction, this paper marks the starting place for future research on models of cellular adhesion and migration as well as the molecular basis for cell-cell binding.

While Holtfreter and Townes were publishing the results of their experimental program, Holtfreter was also collaborating with his close friend and colleague Viktor Hamburger. Hamburger and Holtfreter had both been students in Spemann’s laboratory in Germany. Whereas Holtfreter left Germany for political reasons, Hamburger left as a result of rising anti-Semitism in the 1930s. Hamburger’s research in the United States concentrated on developmental genetics and, later, nerve growth using primarily chicken embryos. Nevertheless, Hamburger worked with Holtfreter to craft a landmark chapter on amphibian development for the 1955 book representing the state of the art, Analysis of Development. Their chapter documented research stemming from the Spemann tradition and provides an invaluable overview of many of the most important advances in experimental embryology in the twentieth century. Although Holtfreter continued to research cell affinities and movement in the aggregating behavior of slime molds after 1956, his essay with Hamburger was his last major scientific publication and in many ways marked the culmination of his research trajectory from Germany.

Holtfreter was the recipient of numerous awards during his career including two Rockefeller fellowships, a Guggenheim fellowship, and Fulbright fellowship. He was elected to the National Academy of Sciences, the American Academy of Arts and Sciences, and the Swedish Academy of Sciences. In 1968 the American Society of Zoologists meeting and the Hahnemann Symposium were both dedicated to Holtfreter. At the University of Rochester he was named Tracy H. Harris Professor in 1966.

Holtfreter continued as an emeritus professor until 1981. During his retirement he returned to painting and travel until he lost his eyesight. In 1992 Holtfreter died at age ninety-one in Rochester.

BIBLIOGRAPHY

Holtfreter’s papers will be donated to the archives at the Marine Biological Laboratories at Woods Hole. His art has been donated to the University of Rochester Art Museum.

WORKS BY HOLTFRETER

With H. Bautzmann, Hans Spemann, and Otto Mangold. “Versuche der Analyse der Induktionsmittel in der Embryonalentwicklung.” Naturwissenschaften 20 (1932): 971–974.

“Differenzungspotenzen isolierter Teile der Anurengastrula.” Roux’ Archive für Entwicklungsmechanik 138 (1938): 657–738.

“Veränderung der Reaktionsweise im alternden isolierten Gastrulaektoderm.” Roux’ Archive für Entwicklungsmechanik 138 (1938): 163–196.

“A Study of the Mechanics of Gastrulation.” Parts 1 and 2. Journal of Experimental Zoology 94 (1943): 261–318; 95 (1944): 171–212.

“Neuralization and Epidermization of Gastrula Ectoderm.” Journal of Experimental Zoology 98 (1945): 169–209.

“Neural Differentiation of Ectoderm through Exposure to Saline Solution.” Journal of Experimental Zoology 95 (1945): 307–340.

With Viktor Hamburger. “Amphibians.” In Analysis of Development, edited by Benjamin H. Willier, Paul A. Weiss, and Viktor Hamburger. New York: W. B. Saunders, 1955. A masterful overview of the experimental embryology of amphibians.

With Philip L. Townes. “Directed Movements and Selective Adhesion of Embryonic Amphibian Cells.” Journal of Experimental Zoology 123 (1955): 53–120.

OTHER SOURCES

Gerhart, John. “Johannes Holtfreter’s Contributions to Ongoing Studies of the Organizer.” Developmental Dynamics 205 (1996): 245–256.

———. “Johannes Holtfreter.” Biographical Memoirs of the National Academy of Science 73 (1998): 3–22.

Gilbert, Scott, ed. A Conceptual History of Modern Developmental Biology. New York: Plenum Press, 1991.

Hamburger, Viktor. The Heritage of Experimental Embryology: Hans Spemann and the Organizer. New York: Oxford University Press, 1988.

Keller, Ray. “Holtfreter Revisited: Unsolved Problems in Amphibian Morphogenesis,” Developmental Dynamics 205 (1996): 257–264.

Steinberg, Malcolm, and Scott Gilbert. “Townes and Holtfreter (1955): Directed Movements and Selective Adhesion of Embryonic Amphibian Cells.” Journal of Experimental Zoology 301A (2004): 701–706.

Michael R. Dietrich