Amphibians 



271 



indication that it has a direct effect, similar 

 to that of the normal neural inductor, rather 

 than a "relay" effect. However, Shen did 

 observe necrotic cells in all of his experi- 

 mental series. Furthermore, as has been 

 pointed out by Needham himself ('39, '42) 

 and by Brachet ('45), some substances, at 

 extremely low concentrations, may be com- 

 paratively more toxic, diffusible, or bio- 

 logically active than others. It appears, there- 

 fore, that the questions of whether poly- 

 cyclic hydrocarbons act directly or indi- 

 rectly and whether such substances are at 

 all involved in normal neural induction 

 cannot yet be answered. 



The same uncertainty seems to apply to 

 fatty acids and nucleic acids. Their neuraliz- 

 ing action was on the whole more pro- 

 nounced than that of steroids. Since such 

 entirely different substances as purified fatty 

 acids from plants (e.g., oleic acid) or ani- 

 mals (e.g., muscle adenylic acid), and com- 

 pletely lipid-free nucleoproteins and nu- 

 cleotides were equally effective, the inves- 

 tigators (Fischer and collaborators, '33, '35; 

 H. Lehmann, '38) refrained from identify- 

 ing any of them with the hypothetical neu- 

 ralizing agent of normal development and 

 ascribed their common effect to an unspec- 

 ified "acid stimulus." Possible cytolytic ef- 

 fects of the substances used have not been 

 considered by these workers, although they 

 have been demonstrated (Holtfreter, '45b; 

 Brachet, '49). A relay-effect of the "acid 

 stimulus" is therefore within the range of 

 possibility. 



FRACTIONATION OF TISSUE EXTRACTS 



One might hope to break the deadlock by 

 comparing the activities of various tissue 

 extracts, obtained by different fractionation 

 methods, with the inductive capacity of the 

 residues. The pioneering experiments were 

 done by Toivonen and Kuusi ('48) and 

 Toivonen ('49, '50), who analyzed the spe- 

 cific inductive capacities of guinea pig liver 

 ("archencephalic inductor") and kidney 

 ("spinocaudal inductor") by testing a great 

 variety of fractions of these tissues obtained 

 by extraction with alcohol and petroleum 

 ether, dialysis, treatment with salt solutions, 

 and heat treatment. All implants were made 

 in whole Triturus embryos. The authors 

 suggest that there exist two qualitatively 

 different inducing substances, an archen- 

 cephalic agent which is dialyzable, thermo- 

 stable and ether-soluble, and a spinocaudal 

 agent which is thermolabile and not ex- 



tractable in ether. Kuusi ('51) continued the 

 analysis of the same tissues, testing tissue 

 homogenates, isolated nuclei, protein frac- 

 tions, cytoplasmic granules, ribonuclease- 

 treated homogenate, formol-treated tissue 

 and others. The capacity for spinocaudal 

 inductions was not associated with cyto- 

 plasmic granules or with extracted nucleo- 

 proteins but seemed to be linked with protein 

 and was easily lost by fractionation proced- 

 ures. The capacity for archencephalic in- 

 ductions seemed to be rather stable. How- 

 ever, no clear-cut chemical separation of 

 the two hypothetical agents has been ob- 

 tained. 



In order to avoid the interference of 

 regional host effects, Yamada (personal com- 

 munication) used isolated gastrula ectoderm 

 as the reacting material. He found that a 

 0.14 M sodium chloride extract (supernatant) 

 of guinea pig kidney induces somites and 

 spinal cord. The extract after heat treatment, 

 and an RNA-protein fraction isolated from 

 the original extract, induced archencephalic 

 structures. DNA-protein of the same tissue 

 gave also archencephalic indvxctions. The re- 

 sults are interpreted in terms of a "dorsaliz- 

 ing" and a "caudalizing" factor. 



Although the techniques employed by 

 these workers were not yet adequate for a 

 chemical characterization of the different 

 agents, this type of experiment seems to be 

 a particularly promising approach to the 

 problem of induction. 



RELATION BETWEEN INDUCTION AND 

 NUCLEIC ACIDS 



Brachet ('45) has marshalled a variety 

 of data in support of his contention that 

 nucleic acids, and particularly RNA found 

 in small granules of the cytoplasm, are 

 responsible for inductions by both normal 

 and abnormal inductors. 



In sectioned amphibian embryos stained 

 with pyronin or toluidine blue, Brachet 

 ('40, '43) foimd an abundance of basophilic 

 cytoplasmic elements whose stainability was 

 lost after treatment with ribonuclease, sug- 

 gesting that the basophilia was due mainly* 

 to the presence of RNA. Microscopic study 

 of the distribution of basophilic elements in 

 amphibian embryos showed a high con- 

 centration in the upper blastoporal lip which 

 decreased during invagination of this mate- 



* Toluidine blue, unless applied within a strongly 

 acid range, stains proteins in addition to nucleic acid 

 (Herrmann, Nicholas, and Boricious, '50). Brachet 

 does not mention having controlled the pH. 



