272 



Embryogenesis: Progressive Differentiation 



rial.* A simultaneous increase in stainable 

 elements in the medullary plate suggested 

 a transfer of RNA from the inductor to the 

 neural plate. Later, basophilia was again 

 strong in the notochord, decreasing in the 

 mesoderm in a ventral direction, but the 

 epidermis contained these granules as well.f 

 Exact quantitative determinations of this dis- 

 tribvxtion pattern have not been made. 



Using the ultracentrifuge, Brachet and co- 

 workers ('40, '42, '44) isolated from homog- 

 enized embryonic and adult tissues small 

 cytoplasmic granules which contained all or 

 most of the RNA, besides phosphatides, pro- 

 teins with — SH groups, and certain enzymes 

 which varied with the type of tissue. These 

 granules which were assumed to be identi- 

 cal with the basophilic elements in sections 

 proved to be inductive when applied as coag- 

 ulated grafts, as was also tobacco mosaic 

 virus. Treatment of organizer tissue, adult 

 organs, or tobacco mosaic virus with ribo- 

 nuclease resulted in a conspicuous decrease 

 or loss of their inductive capacity. All these 

 data suggested that either RNA or its nu- 

 cleotides are instrumental in normal and 

 experimental neuralization. 



Persuasive as these data may appear, they 

 are subject to several objections which re- 

 duce their conclusiveness. (1) The basophilic 

 elements of the amphibian embryo occur 

 over much wider ranges of tissues and de- 

 velopmental stages than are normally in- 

 volved in induction. Their distribution seems 

 to reflect rates of general metabolic activity 

 rather than inductive activity. (2) The 

 scanty histological evidence offered by 

 Brachet ('45) indicates that the implanted 

 granules are much less effective inductors 

 than are whole tissues. Kuusi ('51) found 

 that the isolated granules from guinea pig 

 liver and kidney are not more potent than 

 are the cell residues, or the supernatant of 

 the homogenates. Contrary to Brachet, 

 Kuusi did not find any relationship between 

 the RNA content of the various preparations 



* Pasteels ('49b) found a steady increase of baso- 

 philic granules in the prechordal mesoderm. 



t In late gastrulae and neurulae of T. alpestris, 

 Cagianut ('49) found pyronin-stainable cell struc- 

 tures distributed in both the neural plate and the 

 archenteron roof along a cephalocaudal gradient 

 and diminishing in density toward the ventral 

 regions of the ectoderm and mesoderm. These struc- 

 tures consisted mainly of perinuclear and cortical 

 caps, and of long filaments such as Brachet ('40, 

 '43) has already described. It appears rather hazard- 

 ous to identify these gross structures with the gran- 

 ules of various sizes that have been isolated from 

 centrifuged homogenates. 



and their inductive capacity. The results ob- 

 tained with tissues that had been hydrolyzed 

 with crystalline ribonuclease led Kuusi to 

 the conclusion that this treatment fails to 

 reduce their inductivity both in a quantita- 

 tive and in a qualitative sense. (3) Doubts 

 about a correlation between RNA content 

 and inductivity of a graft are strengthened 

 by the observation of Toivonen and Kuusi 

 ('48) that proteolytic enzymes practically in- 

 activate inductive tissues. (4) Different prep- 

 arations of crystalline ribonuclease contain 

 proteolytic enzymes (Cohen, '44; McDon- 

 ald, '48), and it appears that the prepara- 

 tions used by Brachet have not been assayed 

 for such impurities; on the other hand, its 

 efficiency in removing the RNA present in 

 isolated granules or in tobacco mosaic virus 

 has been questioned (Loring, '42; Claude, 

 '44). (5) Observations of Brachet himself 

 ('49) make it likely that the neuralizing 

 activity of pure nucleic acids and nucleotides 

 is due to a relay mechanism because these 

 substances in low concentrations cytolyze 

 ectoderm explants of the axolotl. 



The above studies have the merit of draw- 

 ing attention to the possible significance of 

 cytoplasmic granules in morphogenetic proc- 

 esses, but they do not settle the problem of 

 the chemical nature of the neuralizing agent 

 in favor of nucleic acids or any of the 

 other components of the basophilic elements. 



NEURALIZATION IN RESPONSE TO A 

 TRANSIENT CELL INJURY 



A new approach to the analysis of induc- 

 tion was opened by the discovery of Barth 

 ('41) that the isolated ectoderm of A. 

 punctatum, in contrast to the ectoderm of 

 other species, can form neural structures in 

 the absence of a tangible inductor, namely 

 when reared in standard solution (Fig. 102). 

 Holtfreter ('44b) confirmed this result. He 

 showed that it is correlated with cellular 

 disaggregation of the explant, the degree of 

 disaggregation being proportional to that 

 of subsequent neuralization. Obviously, A. 

 punctatum is more susceptible than other 

 urodeles to ordinary standard solution which 

 hitherto had been considered as "neutral" 

 with regard to factors that direct cell de- 

 termination. One could anticipate that if 

 ectoderm of A. punctatum were cultured in 

 a less injurious medium it would become epi- 

 dermal, and that the ectoderm of other spe- 

 cies, for which standard solution is not 

 injurious, could be neuralized if it would 

 undergo a transient disaggregation. Subse- 



