270 



Embryogenesis: Progressive Differentiation 



ATTEMPTS TO ISOLATE AND IDENTIFY 

 THE INDUCTIVE AGENTS 



Several groups of workers set out to sub- 

 ject embryonic amphibian tissues and in- 

 ductive adult organs to various extraction 

 methods in the hope that the active agents 

 could be isolated and chemically identified. 

 In addition, a large number of well charac- 

 terized chemical compounds were tested. The 

 extracts and svibstances were usually in- 

 cluded in supporting material, such as agar 

 or albumen, which was then grafted into the 

 blastocoele cavity of early urodele gastrulae. 

 Needham ('42) and Brachet ('45) have dis- 

 cussed these experiments extensively. We 

 shall confine ourselves to some pertinent 

 points. 



At the beginning, we wish to emphasize 

 the methodological difficulties involved in 

 this kind of investigation. They arise mainly 

 from the fact that the ectoderm and ento- 

 derm of the gastrula contain neuralizing 

 agents in a masked form which can be 

 liberated or activated by practically any 

 treatment that kills the tissue. The im- 

 planted tissue or substance may be toxic 

 and kill surrounding host cells which, in 

 turn, would release their liberated agents 

 into the adjacent, non-injiu'ed ectoderm 

 (Barth and Graff, '38; Okada, '38; "relay 

 mechanism," Holtfreter, '45b). Furthermore, 

 as Brachet points out, implanted dead or 

 adult tissue is subject to degradations by the 

 enzyme systems of the host. For these reasons, 

 it is impossible, in principle, to obtain conclu- 

 sive direct evidence that any of the tissues or 

 substances which were implanted contains, 

 or is identical with, the neuralizing agent of 

 the living archenteron roof. 



The following "inductors" operate prob- 

 ably indirectly ("relay"), by cytolyzing host 

 tissue: inorganic substances, such as silica 

 and kaolin (Okada, '38), cephalin and digito- 

 nin (Barth and Graff, '38; Barth, '39). Like- 

 wise methylene blue, claimed by Wadding- 

 ton, Needham and Brachet ('36) and by 

 Beatty, deJong and Zielinski ('39) to have 

 neuralizing effects, was found to be effective 

 only in toxic concentrations (Holtfreter, 

 '45b).* This was true also of sulfhydryl 

 compounds for which Brachet and Rapkine 

 ('39) claimed a neuralizing activity. As to 

 the inductivity of thiocyanate (Ranzi and 

 Tamini, '39), the evidence suggests that the 

 observed neuralizations were either due to 

 a toxicity of the chemical or to contamination 

 of the ectoderm with mesoderm. The much- 



* Pasteels ('51) reports that data of his own do 

 not support the latter conclusion. 



discussed question whether or not glycogen 

 can induce is still open; very likely, the posi- 

 tive cases reported can be attributed to im- 

 purities of some glycogen preparations. 



The Cambridge workers contended that 

 the naturally occurring neural inductor 

 ("evocator") is a steroid compound. This 

 claim was based on the effectiveness of the 

 unsaponifiable fraction of ether extracts from 

 embryos and adult tissues, and further- 

 more, on the effectiveness of a variety of 

 chemically identified steroids (see Fig. 105; 

 for references, see Waddington, '40; Need- 

 ham, '42). Concerning the first point, the 

 evidence is not very striking with respect to 

 the frequency and the histological type of 

 inductions observed. Unfortunately, control 

 experiments showed that coagulated egg al- 

 bumen, which usually served as carrier, 

 could by itself induce similar palisade struc- 

 tures and neuroid cell aggregations, which 

 further weakens the case. Fischer ('35) and 

 H. Lehmann ('38) found the unsaponifiable 

 fraction to be inactive when freed from 

 acids and salts, but the saponifiable fraction, 

 containing fatty acids, to be strongly active. 

 This was confirmed by Barth and Graff 

 ('38) and Toivonen ('49, '50), who found, 

 furthermore, that the extracted residues were 

 far more active than their ether extracts. 

 The second point, the effectiveness of chem- 

 ically identified polycyclic hydrocarbons, 

 most of which had been prepared syn- 

 thetically (Waddington, '38a), deserves more 

 serious consideration. Some of these com- 

 pounds are known as estrogenic, others as 

 carcinogenic agents; however, some other- 

 wise biologically inactive substances did in- 

 duce as well, and there was no parallelism 

 between the neurogenic and the estrogenic 

 or carcinogenic activity of these compounds. 



Experiments of Shen ('39) with a water- 

 soluble polycyclic hydrocarbon (a carcino- 

 gen) have been used as the main argument 

 in support of the steroid nature of the normal 

 neural evocator. The substance, in very 

 low doses, was mixed with crystalline al- 

 bumen which was then implanted into the 

 blastocoele. The alleged correlation between 

 dosage and percentage of inductions is not 

 clear: a peak for neural inductions seems to 

 be at a concentration of 0.0125y,-|- while the 

 percentage of neuroid structures simply in- 

 creases with decreasing dosage. 



The effectiveness of very low concentra- 

 tions of this hydrocarbon was taken by 

 Waddington ('40) and Needham ('42) as an 



fThis peak has statistically little significance 

 (chi square test). 



