Early Attempts to Locate the Organizer Molecules
A Selective History of Induction III
The mechanism of this induction was controversial from the outset. Spemann had favored the assimilatory induction through the ectoderm, but evidence was mounting that the underlying mesoderm was critical. The article by Bautzmann et al. (1; actually a compilation of experiments done separately) showed that not only would dead organizer tissue induce neural plates, but so would dead intestine and epidermis. [Indeed, the non-specificity of induction caused enormous concern. One possibility is that several of these tissues contain follistatin (2). Salome Gluecksohn Waelsch (3) has claimed that Else Wehmeier was actually the first person who observed that dead organizer tissue still induced the competent ectoderm]. In that compilation, Johannes Holtfreter demonstrated that when ectoderm is wrapped around the dorsal mesoderm would form brain structures. Holtfreter (4) followed this up with his exogastrulation studies wherein the dorsal mesoderm failed to make contact with the overlying ectoderm. In these instances, the ectoderm did not acquire a neural character, again suggesting that the inducing signal appeared to be transmitted vertically from the mesoderm to the ectoderm.
The search for the "organizer molecule" was one of the first worldwide biological research programs. Researchers from Germany, the United Kingdom, the United States, the Netherlands, Japan, Finland, Belgium, and China all sort this elusive entity. Harrison (quoted in 5) referred to the amphibian gastrula as "a new Yukon to which eager miners were now rushing to dig for gold around the blastopore." Løvtrup and his colleagues (6) remarked that "few compounds, other than the philosopher's stone, have been searched for more intensely than the presumed agent of primary induction in the amphibian embryo." In 1961, Lauri Saxon (7) demonstrated that neural induction could occur through a 150 micron thick, 0.8 micron pore size filter, strongly suggesting that the inducer was diffusible. The biochemical purification of this Induktionsstoffe had been part of the Finnish laboratory's program, starting with Toivonen's student, Taina Kuusi (8) and became the focus of the ongoing Tiedmann' research program (9).
Others were trying to find the inducer by seeing which natural substances could induce neural plate formation when added to competent ectoderm or implanted into the blastocoel. One of the laboratories testing such compounds was that of Joseph and Dorothy Needham and C. H. Waddington. Joseph Needham was trying to create a biochemical embryology that would synthesize the reductionism of chemists with the wholism of biologists (see 10). In their work in 1933 and 1935, Waddington and the Needhams (11, 12) showed that the ether extracts of adult newts could act as an organizer. Since this activity could turn presumptive epidermis into non-specific neural tissue, Waddington called this activity as the "evocator." (The molecules specifying the type of neural tissue were referred to as the "individuators." The properties of the evocator fraction suggested that it was a steroid, and both natural and artificial steroids were found to induce neural plates. A sterol inducer made a great deal of sense (13), since sterols had been found to be the basis for male and female sex hormones, cancer-producing hydrocarbons, cardiac glycosides, and vitamin D. Moreover, Emil Witschi had evidence that eggs stored these compounds. However, sterols weren't the only chemicals that induced neural development. They also found that methylene blue and other synthetic molecules could also induce the ectoderm to become neural. In 1936, Waddington, Needham, and Jean Brachet (14) hypothesized that the evocator substance was produced throughout the embryo, but it was just released or activated in one particular region. This fit in well with Holtfreter's (15) discovery that non-inducing regions of the amphibian gastrula could acquire the ability to induce when they were killed.
Figure 1 Joseph and Dorothy Needham and the evidence that hydrocarbons could induce neural tube formation. This was to be the foundations of biochemical embryology. (The Needhams soon afterwards went to China, where Joseph Needham became the foremost Western authority on Chinese science).
Figure 2 Johannes Holtfreter and the experiments that indicated the notochord was producing Organizer molecules.
The search for the Organizer turned out to become extremely frustrating, with some biologists declaring that maybe there were no organizer molecules (see 16). It turned out, though, that the biochemical tools to isolate proteins and small molecules were not sufficiently refined. By the late 1980s, several investigators felt that molecular biology finally had something to offer them. Fred Wilt (17) urged that the time had come for molecular biology to try something more challenging—like explaining development. That same year, John Gurdon (18) also saw the times changing. After reviewing the highlights of research on neural induction, he concluded, "Nucleic acid technology has probably now reached a sufficient level of precision and efficiency of operation to be usefully applied to the analysis of inductive responses..." During the past decade the techniques of molecular biology has identified several soluble molecules—Follistatin, Chordin, Noggin, Xenopus nodal-related-3, and Cerberus—that have organizer function (see 19).
Literature Cited
1. Bautzmann, H., Holtfreter, J., Spemann, H., and Mangold, O. 1932. Versuche zur Analyse der Induktionsmittel in der Embryonalentwicklung. Naturwiss. 20: 971-974.
2. Ritvos, O., Tuuri, T., Erämaa, M., Sainio, K., Hilden, K., Saxén, L., and Gilbert, S. F. (1995). Activin disrupts epithelial branching morphogenesis in developing murine kidney, pancreas, and salivary gland. Mechanisms of Development 50: 229-245.
3. Waelsch, S. G. (a.k.a. Gluecksohn-Schoenheimer) 1992. The causal analysis of development in the past half century: a personal history. Development (suppl.) 1992: 1-5.
4. Holtfreter, J. 1933a. Die totale Exogastrulation, eine Selbstablösung des Ektoderms vom Entomesoderm. Roux' Arch. f. Entw. mech. 129: 669-793.
5. Twitty, V. C. 1966. Of Scientists and Salamanders. Freeman, San Francisco. p. 39.
6. Løvtrup, S., Landström, U., and Løvtrup-Rein, H. 1978. Polarity, cell differentiation, and primary induction in the amphibian embryo. Biol. Rev. 53: 42.
7. Saxén, L. 1961. Transfilter neural induction of amphibian ectoderm. Dev. Biol. 3:140-152.
8. Kuusi, T. 1951. Über die chemische Natur der Induktionsstoffe im implantatversuch bei Triton. Experientia 7: 299 -300.
9. Tiedmann, H. and Tiedmann, Ha. 1964. Das Induktionsvermögen gereinigter Induktionsfaktoren im Kombinationsversuch. Rev. Suisse de Zool. 71: 117-137.
10. Abir-Am, P. 1991. The philosophical background of Joseph Needham's work in chemical embryology. In Gilbert, (ref. 5), pp. 159-180.
11. Waddington, C. H. and Needham, D. M. 1935. Studies on the nature of the amphibian organization centre II. Induction by synthetic polycyclic hydrocarbons. Proc. Roy. Soc. (Lond.) B 117: 310-317.
12. Waddington, C. H., Needham, J., Nowinski, W. W., and Lemberg, R. 1935. Studies on the nature of the amphibian organization center. I. Chemical properties of the evocator. Proc. Roy. Soc. (Lond.) B 117: 289-310.
13. Needham, J. 1936. Order and Life. Yale University Press, New Haven.
14. Waddington, C. H., Needham, J., and Brachet, J. 1936. Studies on the nature of the amphibian organization centre III. The activation of the evocator. Proc. Roy. Soc. (Lond.) B: 120: 173-198.
15. Holtfreter, J. 1933b. Nachweis der Induktionsfähigkeit abgetöteter Keimteile. Roux' Arch. f. Entw. mech. 128: 584-633.
16. Saxén, L. and Toivonen, S. 1962. Primary Embryonic Induction. Prentice-Hall, NJ.
17. Wilt, F. H. 1987. Determination and morphogenesis in the sea urchin. Development 100: 559-575.
18. Gurdon, J. B. (1987). Embryonic induction—Molecular prospects. Development 99: 285-306.
19. Gilbert, S. F. 1997. Developmental Biology. Fifth edition. Sinauer Associates, Sunderland, MA.