A Selective History of Induction IV
Regional specificity
When induced by non-specific evocators, anterior neural tissue usually forms (Nieuwkoop, 1). Thus, some other factors are needed (Waddington's "individuators") to specify the regionality of forebrain, midbrain, hindbrain, and spinal cord. Otto Mangold (2) showed that the regional position of the blastopore lip-derived dorsal mesoderm determined the anterior-posterior specification of the neural tube. When transplanted into the blastocoel of early gastrulae, the anteriormost mesoderm induced cement glands and eyes, posterior-most mesoderm induced tails, and the intermediate mesoderm induced the appropriate intermediate structures. This could also be seen by transplanting the tissue at the time when it comprised the dorsal blastopore lip (3). Early dorsal blastopore lips gave rise to head structures; late dorsal blastopore lips induced new tails. To further study the organizer phenomenon, Holtfreter (3a) made a blastopore "sandwich," enclosing the dorsal blastopore lip between slices of undifferentiated ectoderm. The blastopore lips from the early gastrula stages induced the most archencephalic (anterior) structures, whereas the blastopore lips of later embryos caused the differentiation of the more posterior neural elements.
Two major sets of models were generated to explain these results. Both involved the notion of gradients. Here we have to digress briefly to discuss the concept of gradients in embryology. Until they were resurrected in the laboratory of Christiane Nüsslein-Volhard, "gradients" had been a "dirty word" for decades (4). There are several reasons for this, one of which was that they were ridiculed by a person who had been one of the leading investigators of such gradients, Thomas Hunt Morgan (5). The introduction of gradients into embryology is often credited to Boveri (6), who did introduce the term "Gefälle" (gradient) into his work. However, as Sander (7) has made explicit, Boveri's concept of a gradient is not the one that we have today. His is more like a step-gradient, wherein the cell membrane separating two cells also separates two concentrations of the same substance. His illustrations, however, show a graded character from the vegetal to the animal pole, and this makes it appear that there are threshold-related gradients in these embryos.
The threshold gradient idea was brought about primarily by Rünnstrom (8) and Hörstadius (9) in their studies of sea urchins. Their experiments indicated that a gradient of vegetal substance was found throughout the embryo, concentrated in the vegetal-most micromeres, and there was conversely a gradient of animalizing factor in the otherdirection. Their countryman T. Gustafson (10) represented this graphically as two intersecting gradients. Gavin de Beer and Julian Huxley (11), along with C.M. Child (12, 13), created the idea of a "gradient field" Here, a morphogenetic field existed that depended upon a system of induction (see 14). However, the cells responded to the inducer in a graded manner depending upon the inducer concentration. In that way, polarity could be generated. This was a very controversial idea. Morgan detested it because it postulated an alternative mode of inheritance (as in planaria dividing). Spemann (15) also did not agree with it, saying that while a streamof water could drive a water mill, the actual mountain cannot. In 1953, Leopold von Ubisch (16) would counter this argument with an example of the mountain cutting through a temperature gradient so that different types of vegetation would be found at different altitudes. Different genes could be activated by different amounts of substance. However, this remained strictly a hypothesis (for more detailed information, see 17).
Numerous investigators attempted to use gradients to explain the results of Mangold's experiments. The quantitative models postulated a gradient of a single molecule that would be formed within the dorsal mesoderm. The amount of this molecule would specify the regional differences in the overlying ectoderm. For instance, Dalcq and Pasteels (18) and Yamada (19) proposed that two intial substances at either end of the mesoderm would interact to produce a gradient of the actual morphogenetic substance (called "organicine" by the Belgian group). The fate of the overlying ectoderm was determined by the amount of organicine in the mesoderm. Quantitative models, such as that hypothesized by Lehmann (20), postulated that the dorsal mesoderm elaborated different substances at its anterior and posterior ends. The differences in the ratios of these substances produced by these interpenetrating gradients determined the type of neural tissue.
Evidence for the dual gradient model was obtained by Sulo Toivonen. Toivonen's mentor, Gunnar Ekman had worked in the laboratory of H. Braus, the close friend of Spemann who had given Spemann the double assurance idea. After Braus' death in 1924, Ekman spent some time in Spemann's laboratory in Freiburg. Ekmann was intrigued with Holtfreter's experiments on lens induction, and suggested that Toivonen find a lens-specific inducer (21). Toivonen's (22) research did not yield a lens inducer. However, he did find specific neural inducers. Adult guinea pig liver, for instance, only induced spinal cord and did not induce head structures. Conversely, the guinea pig bone marrow was an excellent inducer of heads, but did not induce the trunk neural tube. Similar results were also forthcoming from one of Holtfreter's graduate students, Siao-Hui Chuang (23), but whereas Chuang focused on whether the host environment might alter the induction (it did not), Toivonen was interested in the type of induction, "Leistungspezifitt", produced by these tissues. Moreover, instead of merely recognizing two specificities (head vs trunk) , he divided the neural axis into four regions: anterior (archencephalic) head structures, posterior (deuterencephalic) head structures, trunk structures, and tail structures. Needham (24) called this work of Toivonen's and Chuang's the third fundamental discovery about the organizer. (The first two were the discoveries that killed organizers still had inducing capacity and that killing non-inducing tissues could convert them into inducing ones).
Figure 1 Sulo Toivonen
The test of this hypothesis was accomplished by Toivonen and his student, Lauri SaxEn. This partnership, as Hamburger (21) has noted, "left its mark on experimental embryology to this day." Toivonen and SaxEn (25, 26) placed an archencephalic inducer, guinea pig liver, together with a spinocaudal (mesodermal) agent, guinea pig bone marrow, into blastocoels or between sheets of competent ectoderm. In addition to inducing tail and archencephalic structures, the dual implants also formed the intermediate spinal cord structures that neither did alone. Moreover, in the Einsteck experiments, the induced structures were arranged along an axis from archencephalic to tail. Toivonen and SaxEn interpreted these results in terms of two gradients, each arising from its specific inducer. These gradients were projected upon the competent ectoderm. The neuralizing gradient was highest dorsally in the embryo, evenly distributed along the anterior-posterior axis. The mesodermalizing gradient (one must recall that the posterior end of the neural tube becomes mesoderm) is highest in the dorsal midline, extending only into the deuterencephalic area. Needham (24) reflected that this work "is assuredly the fourth fundamental experiment in this field."
Figure 2 The Saxén-Toivonen model of regional specificity. The neural inducer is highest dorsally throughout the neurula. The caudalizing "mesodermal" factor is highest in the posterior of the neurula, tapering off anteriorly.
Meanwhile Petr Nieuwkoop and his laboratory (1) in the Netherlands had revised the Waddington's (27) evocator/individuator and provided evidence for a two-step process of neural induction. The initial "activation" would induce competent ectoderm to become archencephalic structures. These neurally specified cells would secondarily be "transformed" into more caudal structures by some other factor. Nieuwkoop showed that this caudalizing factor existed in a gradient. Folds of competent ectoderm were implanted at various regions along the dorsal anterior-posterior axis. The distalmost region of each fold remained undifferentiated. The proximal region of each fold showed the specificities characteristic of the region wherein it was inserted into the host. The region between them developed more anterior specification than the region of insertion.
Confirmation of this two-step model was obtained by SaxEn, Toivonen, and T. Vainio (28). They induced competent ectoderm with either a forebrain inducer or a spinocaudal (mesodermalizing) inducer. They then separated the ectoderm from the inducers. And disaggregated the cells. The cells were then reaggregated as pellets. The pellet produced by the forebrain-induced cells gave rise primarily to forebrain structures, while the ectoderm reaggregated from spinocaudal-induced cells gave rise primarily to spinal cord. However, when the two groups of cells were aggregated together, the result was a preponderance of hindbrain structures, structures that neither group of cells would form well on their own.
Figure 3 Evidence for a two-step process of induction. Competent ectoderm was incubated 24 hours with either a forebrain inducer or a spinocaudal inducer. The inducers were then removed and the ectoderm disaggregated. The ectodermal cells were reaggregated either separately or after complete mixing. When the ectoderm that had been induced by forebrain inducers was mixed with ectoderm that had been induced by spinocaudal inducers, the cells formed hindbrain as well as forebrain and spinocaudal structures. Thus, a neural determination step was followed by interactions between the induced cells . (After reference 28).
However important this might be for a model, these neuralizing (activating) and mesodermalizing (transforming) gradients still had to be shown to exist within the actual neurulating embryo. This was done by Toivonen and Saxén (29) in 1968. The archencephalic regions of the neural plate of newt embryos were dissected out and placed in various proportions with posterior dorsal mesoderm from the same stage embryos. These combinations were then used as inducers. At 10:1 and 5:1 ratios of archencephalic cells and mesoderm, the neural cells remained forebrain structures (characterized by eyes and nose). However, at 5:2 ratios, hindbrain structures were seen in the explants for the first time, and at 1:1 ratios, spinal cord was observed. In other words, the posterior mesoderm could take cells that were already committed to become neural and then specify them into more caudal cells. The more the mesodermal portion of the ratio climbed, the more caudal the neural specification.
Recent advances using the techniques of molecular biology have refined the double gradient model. The neural inducers (such as Follistatin, Chordin, and Noggin) appear to induce an anterior (archencephalic) type of neural tissue. This appears to be posteriorized by a gradient of chemical coming from the posterior end. The posteriorizing chemical has been postulated to be a fibroblast growth factor, a Wnt protein, or a retinoid (all seem to posteriorize the neural tissue, and there is evidence for a posterior-to-anterior gradient of at least retinoic acid and eFGF (see 30).
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