A Selective History of Induction II
Spemann's induction experiments
A. Spemann's Lens Induction Experiments
In 1901, Curt Herbst wrote that he thought it possible "to establish the occurrence of formative stimuli which are exerted from one part of the embryo to another, and to demonstrate eventually the possiblity of a complete resolution of the entire ontogenesis into a sequence of such inductions." This prediction of inductive cascades was a bold statement, given that no sequence of events had yet been observed (1, 2).
But that was soon to change. That same year, Hans Spemann (3) published "one of the most significant and seminal papers in the history of embryology" (4), his experimental analysis of lens formation in the frog. Spemann cauterized the prospective retina anlagen in the neurula stage Rana fusca. A few days later, Spemann observed that both the eye and the lens were missing on the operated side of the tadpole. In those cases where the retinal rudiment had not been totally destroyed, the ability to form lenses appeared to correlate with the ability of the remnant to contact the overlying ectoderm. Spemann claimed that contact of the optic vesicle with the overlying ectoderm was needed to turn that ectoerm into a lens, but he did not know whether or not it was a sufficient cause. Moreover, he did not know yet whether the optic cup instructed the ectoderm to form a lens or merely acted as a trigger to permit a pre-existing potency to become expressed. Such certainty would come when he was able to move an optic cup into an ectopic location and observe whether lenses had been formed. He admitted that these transplantations were difficult to perform and that he was not having much success. Spemann's paper was exciting for several reasons, not the least being that he showed it possible to tease out the relationships between embryonic parts. As we know, Spemann was challenged by Ernst Mencl (who claimed to get free, unattached, lenses when he did Spemann's experiment) and was supported by Warren Lewis (who had performed the ectopic grafting experiments that Spemann was trying and claimed that the optic cup could cause ectopic lens formation). Spemann's and Lewis' studies were challenged again by Helen King (5; see 3, 6) who had repeated Spemann's ablation experiments on Rana palustris (a related American frog) and got the opposite results: free lenses, which were not connected to an eye. King's paper sent Spemann back to the laboratory where he did the identical experiment on different species of frog neurulae. He confirmed both his earlier results and Helen King's results (7). There was a component of induction that was species-specific.
There appeared to be two ways to make a lens: induction or self-determination. Spemann put this result in the context of a model proposed the previous year, concerning the operculum of amphibian tadpoles. When the limbs of amphibian tadpoles arise during metamorphosis, they emerge through an opening called the operculum. It had been thought that the limbs mechanically pushed through this barrier. However, when Herman Braus removed the forelimb rudiments, the operculum still opened at the appropriate time. Braus explained this phenomenon by introducing the engineering term "double assurance" into embryology (6). Spemann (8) maintained that double assurance also worked for amphibian lenses, and that while some frogs had only one mechanism of lens determination, most species were in between and had some self-differentiation but still required contact for optimum lens formation.
Figure 1 Hans Spemann and the lens ablation experiment
B. Spemann and Mangold: The Organizer
In 1914, Spemann was made head of the Division of Developmental Mechanics at the Kaiser Wilhelm Institute in Dahlem. He redirected his research away from the formation of specific organs (eye, ear, liver) towards looking at the problem of the earliest stages of embryonic determination. He decided to test the state of determination of the early salamander gastrula, and to that end, he reciprocally transplanted small regions of embryos from one region on one salamander gastrula to a new region on another gastrula. The gastrulae were from differently pigmented specimen of the same species. He would then observe whether the transplanted piece differentiated according to its original source or according to its new environment. In his 1918 paper (9), he concluded that the determination of the neural plate occurs during gastrulation. In addition, he noted the convergent extension of the posterior neural plate.
There was only one exception to the general rule that the transplanted pieces from early gastrulae were not yet determined. When he had transplanted tissue from the region above the dorsal blastopore lip, those cells formed a second neural tube underlain by a notochord and flanked by two rows of somites. One might have expected Spemann to conclude that the dorsal blastopore lip had formed the notochord and that this induced the neural plate. After all, in 1903, Spemann (10) had shown that in constricted eggs, two neural axes form and that the extent of the neural tube appeared to be correlated with the extent to which the underlying archenteron roof mesoderm had progressed. Spemann (10; quoted in 6) had even written that "It is conceivable that the neural plate is induced by the archenteron." However, Spemann was not thinking in those terms in 1918. Rather, he mistakenly held that the upper layer of the dorsal blastopore lip gave rise to neural ectoderm (rather than involuting to form chordamesoderm). Thus, Spemann contended that the dorsal blastopore lip self-differentiated to form neural ectoderm and that this initiated a wave of assimilative differentiation (Andifferenzierung), spreading anteriorly in the plane of the ectoderm (see 6). Spemann's error was pointed out to him by anatomist Hans Petersen of the University of Heidelberg, and by Spemann's new heteroplastic transplants where invaginated cells could be more easily identified. Spemann (11) therefore reinterpreted his dorsal blastopore lip experiment, and he felt that there was a need to do heteroplastic transplants of the dorsal blastopore lip region. In the spring of 1921, he assigned the task to a graduate student, Hilde Proescholdt.
In 1924, Spemann and Proescholdt (now Mangold) published some of their findings on the dorsal blastopore lip transplantations. They had found (a) that the dorsal blastopore lip transplants invaginated almost completely, (b) that the transplanted tissue caused the formation of a secondary neural plate composed almost entirely of host tissue, and (c) that while the notochord was primarily derived from donor tissue, the flanking mesoderm was a combination of donor and host cells. Some somites were chimeric, some completely host, some completely donor. Actually, the published account (12), displaying the terms induction and organizer prominently in its title, is based on only the first two years of research, those encompassing the 1921 and 1922 breeding seasons. Although this publication only refers to six cases, Sander (13) and Fssler (13a) have shown that of the total 275 chimeras, fifty-five survived the operation, of which twenty-eight had prominent secondary neural axes, eleven of which had mesodermal segmentation.
Figure 2 The Spemann and Mangold paper
The term "Organizer" was coined to emphasize the ability of this dorsal blastopore lip tissue to direct the development of the host tissue and to give these redirected cells a coherent, unified, organization. "A piece of the upper blastoporal lip of an amphibian embryo undergoing gastrulation exerts an organizing effect on its environment in such a way that, if transplanted to an indifferent region of another embryo, it causes there the formation of a secondary embryonic anlage. Such a piece can therefore be designated as an organizer." This tissue had the ability to invaginate and differentiate autonomously, to induce the neural plate and, by assimilative induction, to organize somites from the lateral plate mesoderm of the host.
Interestingly, it was this assimilative induction that Spemann and Mangold focused on in the discussion of their paper. The two critical cases where the entire neural plate was composed of host cells went unmentioned in their discussion section (6). The interpretation became different only after Spemann and Otto Mangold devised the Einsteck method of transplantation, wherein a transplant could be slipped into the blastocoel of the gastrula through a small slit in the upper hemisphere. During the ensuing gastrulation movements, the blastocoel narrows, and the transplant adheres to the inner layer of the ventrolateral ectoderm. If it has neural inducing ability, it should induce a secondary neural plate. Spemann's student Bruno Geinitz (14) showed that dorsal blastopore lips transplanted into the blastocoel induced excellent secondary neural tubes, and another student, Alfred Marx (15) showed that pure dorsal mesoderm from a late gastrula could induce the neural plate from the ectoderm, while pure ectoderm could not. Spemann now concluded that the dorsal mesoderm was responsible for neural induction.
Figure 3 Spemann and Mangold and the Nobel prize awarded to Spemann. (Mangold died the year of her publication. Nobel prizes are not given posthumously.)
Literature Cited
1. Herbst, C. 1901. Formative Reize in der tierischen Ontogenese. Ein Beitrag zum Verständnis der tierischen Embryonalentwicklung. Georgi, Leipzig.
2. Gilbert, S. F. 1997. Developmental Biology. Fifth edition. Sinauer Associates, Inc., Sunderland, MA.
3. Spemann, H. 1901. Über Correlationen in der Entwicklung des Auges. Verhand. Anat. Ges. 15: 61-79.
4. Saha, M. 1991. Spemann seen through a lens. In Gilbert, S. F. (ed.) 1991. A Conceptual History of Modern Embryology. Plenum Press, NY. p. 91-108.
5. King, H. D. 1905. Experimental studies on the eye of the frog embryo. Arch. Ent. Mech. 19: 85-107.
6. Hamburger, V. 1988. The Heritage of Experimental Embryology: Hans Spemann and the Organizer. Oxford University Press, Oxford.
7. Spemann, H. 1907a. Neue Tatsachen zum Linsenproblemen. Zool. Anz. 31: 379-386.
8. Spemann, H. 1907b. Zum Problem der Correlation in der tierischen Entwicklung. Verhadl. deutsche zool. Gesell. 17: 22-49.
9. Spemann, H. 1918. Über die Determination der ersten Organanlagen des Amphibienembryonen. Zool. Jahr. Supp. 15: 1-48.
10. Spemann, H. 1903. Entwicklungsphysiologische Studien am Tritonei III. Roux Arch. f. Entw. Mech. 16: 551-631.
11. Spemann, H. 1921. Über den Anteil von Implantat und Wirtskeim an der Orientierung und Beschaffenheit der Induzierten Embryonalanlage. Arch. Entw. mech. Org. 123: 389-517.
12. Spemann, H. and Mangold, H. 1924. Über Induktion von Embryonanlagen durch Implantation artfremder Organisatoren. Roux' Arch. f. Entw. mech. 100: 599-638.
13. Sander, K. 1993. Hans Spemann, Hilde Mangold und der "Organisatoreffekt" in der Embryonalentwicklung. Akademie-Jour. (Jan. 1993): 7-10.
13a. Fässler, P. E. 1996. Hans Spemann (1869-1941) and the Freiburg school of embryology. Internat. J. Devel. Biol. 40: 49-57.
14. Geinitz, B. 1925. Embryonale Transplantation zwischen Urodelen und Anuren. Roux' Arch. f. Entw. mech. 106: 357-408.
15. Marx, A. 1925. Experimentelle Untersuchungen zur Frage der Determination der Medullarplatte. Roux' Arch. f. Entw. mech. 105: 20-44.