304 MOLECULAR MECHANISMS OF DIFFERENTIATION 5 



was investigated. Preliminary measurements indicate that the amount of labelled 

 glycine taken up does not change on addition of the leucine analog. However, the 

 total protein glycine content of the somites after explantation with the analog 

 was found to be smaller than in the controls. This may be regarded as an indi- 

 cation of an enhanced protein breakdown and parallels the observations made 

 with aminoketones discussed previously. Ethionine was found to be without gross 

 effect on the development of explanted embryos under the conditions of cultivation 

 used in these experiments. However, it inhibited the incorporation of tracer glycine 

 into the embryonic proteins (11-13 somite stage) by about twenty per cent. In 

 these cases the effect of the analogs on morphogenesis is not correlated to the 

 inhibition of amino acid incorporation into the embryonic proteins (Schultz and 

 Herrmann, 1958). As a second alternative, it could be assumed that during 

 embryogenesis some tissues or cells depend to a larger extent upon protein 

 formation from large molecular precursors than others and, therefore, are less 

 sensitive to amino acid analogs. Also, it should be considered that different analogs 

 will be incorporated as such into some proteins, as has been observed in the case 

 of ethionine (Levine and Tarver, 195 1). In this way, certain proteins would be 

 altered more than others, thus producing the specific effects. Preliminary ex- 

 periments exploring this alternative did not provide evidence for the presence 

 of leucine analog in paper chromatograms obtained from protein hydrolyzates 

 of either early embryos or of muscle tissue from older animals. 



A series of studies on the effect of amino acid analogs on the early development 

 of amphibian and chick embryos were also carried out by Waddington and Sirlin 

 (1954) and Feldman and Waddington (1955). The embryos in situ were exposed 

 to solutions of labelled amino acids and amino acid analogs and an evaluation of 

 the uptake of labelled material was attempted by radioautographs' of the embryos 

 (the number of silver grains was determined as an index of the incorporation 

 rate). In other experiments the protein activity was also determined by a con- 

 ventional Geiger counting procedure. Xenopus embryos were kept for 21 hours 

 in a solution of glycine- i-*'*C. It was shown radioautographically that a distinct 

 maximum of radioactivity occurred in the epidermis and in the somites. Notochord 

 and neural tube showed only one-half of the activity and the endoderm only 

 slightly more than one tenth. Measurements obtained by counting techniques 

 demonstrated that the incorporation of glycine into whole amphibian embryos was 

 decreased to about one-half when the analog para-fluorophenylalanine was 

 added. 



A similar study was carried out with early chick embryos between stages seven 

 and nine, corresponding to the formation of the first somites. Evaluation of radio- 

 autographs seemed to indicate that the neural fold showed the highest incorporation 

 of labelled methionine-^^S. In the early stages, the ectoderm seemed to be more 

 active than the axial mesoderm and the reverse condition prevailed in the later 

 stages. However, the differences observed in chick embryos were much smaller 



' Rates of protein synthesis can, at best, be derived from time curves for the specific activities 

 of tissue proteins. Since radioautographs do not lend themselves readily to the establish- 

 ment of such curves their value for measurements of rates of protein synthesis is limited at 

 the present time. 



