256 CELLS, TISSUES, AND ORGANISMS 



or haploid condition per se but to the degree of morphogenesis attained 

 by diploid and haploid organs ( Brachet, 1944) . 



In summary, all the evidence we have concerning RNA distribu- 

 tion and synthesis in normal and experimental embryos shows that 

 these phenomena are always closely linked to morphogenesis. One 

 should nor forget, however, that RNA is only one of the many con- 

 stituents of ribonucleoprotein particles. There is no proof, for the time 

 being, that RNA in itself is more important for morphogenesis than the 

 proteins and lipids with which it is associated in ergastoplasmic struc- 

 tures. Therefore, experiments designed to demonstrate in an unam- 

 biguous way the role of RNA itself in morphogenetic processes are 

 highly desirable. 



Brachet and Ledoux ( 1955 ) and Brachet ( 1959 ) have attempted 

 to attack this important point in a direct way— by treating living am- 

 phibian eggs with ribonuclease. It was hoped that the enzyme might 

 penetrate into the living cells, inactivate or break down the RNA they 

 contained, and exert important morphogenetic efiFects. As we shall see, 

 these hopes have not been entirely fulfilled, because of the poor pene- 

 tration of ribonuclease into amphibian eggs once cleavage is over. 



It is easy to demonstrate that ribonuclease quickly inhibits cleav- 

 age in amphibian eggs and that the nuclei are usually blocked in inter- 

 phase. However, because the penetration of the enzyme is slow and in- 

 complete, only the blastomeres forming the outer layers of the morula 

 are irreversibly blocked in their development. If the treated morulae 

 are brought back to the normal medium, after a few hours of treatment 

 with ribonuclease, the innermost blastomeres, which surround the 

 blastocele, resume cleavage. They finally migrate tlirough the dying 

 or dead outer blastomeres and form an atypical undifferentiated ecto- 

 derm (Figure 12). If the eggs are treated with a mixture of ribo- 

 nuclease and RNA, or if they are placed in an RNA-containing medium 

 after the ribonuclease treatment, one can occasionally obtain the for- 

 mation of a nervous system (Figure 13); it lies on a bed of large, 

 blocked cells. The fact that we have never yet obtained a nervous sys- 

 tem after treatment with ribonuclease alone, but have obtained several 

 "neurulae" after a ribonuclease-RNA treatment, provides a definite in- 

 dication of a role for RNA in normal induction. 



Penetration of ribonuclease at later stages of development is, as a 

 rule, very poor, and therefore Httle or no effect on morphogenesis is 

 observed. One can, however, occasionally find ribonuclease prepara- 

 tions which are more active than others; since their effects can be 

 duplicated by adding small amounts of versene ( EDTA ) to otherwise 

 inactive preparations of ribonuclease, it is probable that the active 

 preparations contain some chelating agent as a contaminant. 



When blastulae or gastrulae are treated with ribonuclease rein- 



