524 MOLECULAR MECHANISMS OF DIFFERENTIATION 5 



of the developing Distaplia bud has formed lOO cells, mitosis comes to a complete 

 standstill in 6 of these cells and from that time on they begin to develop the 

 morphological characteristics of muscular cytodifferentiation. In the completely 

 developed zooid these 64 muscle cells appear in the form of eight strands of four 

 cells on either side of the thorax. In the remaining mesodermal cells, proliferation 

 ceases at a later stage with subsequent formation of blood and other mesenchymal 

 tissues. Similarly sharp separations of multiplication and cytodifferentiation can be 

 found in almost all cell types of ascidians. 



In higher forms, e.g. vertebrates, an incompatibility of proliferation and differ- 

 entiation was demonstrated more directly in tissue culture experiments. Begin- 

 ning with the experiments of Fischer and Parker (1929) it was found again and 

 again that under conditions of rapid proliferation little differentiation took place, 

 while in slowly growing cultures manifestations of differentiation could be clearly 

 observed. The classical examples are: the pigment formation in slowly growing 

 cells of iris epithelium (Doljanski, 1930- 1931a) or the formation of glycogen 

 granules in slowly growing liver cells (Doljanski, 1930- 1931b). Both of these cell 

 products do not form in rapidly growing cultures. The possibility that apparent 

 oscillations of embryonic growth curves indicate alternating periods of more or 

 less rapid differentiation has been suggested by Schmalhausen (1926) and Schmal- 

 hausen and Stepanova (1926) and is discussed by Needham (1931). However, 

 statistical validity of these oscillations would have to be re-examined. 



A possible relationship of proliferation and differentation in early embryoge- 

 nesis can be explored in the embryos of sea urchins and of amphibians where the 

 two parameters have been defined to some extent in chemical terms. After the 

 initial cleavage stages of sea urchin embryos, an increase in cell number is paral- 

 leled by a corresponding increase in DNA while the RNA and protein contents 

 remain unchanged^ Neither the increase in the quantity of DNA, nor the rate of 

 uptake of -^^P into the RNA molecule indicates a period of declining proliferative 

 activity during development of the larvae (Villee, Lowens, Gordon, Leonard 

 and Rich, 1949). In spite of this uniformity in the rate of proliferation, a distinct 

 period of differentiation can be recognized in the later blastula stage which 

 is indicated by the appearance of mitochondria and of new mitochondrial 

 enzymes (Gustafson and Lenicque, 1952; Gustafson and Lenicque, 1955; Shaver, 

 1956). This phase of difTerentiation coincides with the rapid increase in the in- 

 corporation of amino acids into mitochondrial proteins, a high rate of incorpo- 

 ration observed in the microsomal fractions, and an increase in the utilization 

 of acetate for RNA synthesis (Hultin, 1953d). Thus, the investigations of early 

 sea urchin development do not seem to reveal an incompatibility of proliferation 

 and differentiation with respect to the indicated parameters. 



^ Apparently, total RNA content remains constant, while RNA incorporation increases at 

 certain periods. Determinations of DNA as a measure of proliferation during early phases 

 of development of sea urchin or amphibian embryos have to be carefully evaluated in view 

 of the findings of Hoff-Jorgensen and Zeuthen (1952) and Marshak and Marshak (1953). 

 Also a certain error is introduced by the fact that a change in the rate of mitosis will affect 

 the DNA figures to some extent. 



