528 MOLECULAR MECHANISMS OF DIFFERENTIATION 5 



functional product of this cell. Due to its characteristic absorption spectrum, hemo- 

 globin can be determined qviantitatively in single cells and in this way its accumu- 

 lation can be followed in the maturing erythrocyte at different stages of develop- 

 ment. By measurements of cell number, total cell volume, and protein content per 

 unit cell volume, a comparison could be made between the rate of total protein 

 production (growth) and hemoglobin formation (differentiation). It was found 

 that only in a relatively late phase of erythrocyte development, in the polychromatic 

 erythrocyte, does hemoglobin formation take place. At this stage total protein 

 production has come practically to a standstill and the RNA content of the cells 

 has reached a minimum. These results seemed to indicate that in this cell type a 

 very distinct complementarity of growth and differentiation exists. However, 

 the possibility was considered that perhaps globin, the protein moiety of hemo- 

 globin, was formed at an earlier proliferative phase of erythrocyte development 

 while the formation of the hemin component took place only at the later stage. 

 An attempt was made to decide this question by comparing the incorporation rates 

 of labelled glycine into the hemin and the globin components of the hemoglobin 

 molecule in immature erythrocytes formed after phenylhydrazin hemolysis. 



Although the results of these experiments seem to support this assumption, 

 further extension of these data would add to their conclusiveness'. Even if the inter- 

 pretation is accepted the fact that hemin formation during later development 

 presupposes activation and presumably formation of an enzyme protein re- 

 quired for this synthesis. Again, it may be that increased turnover of at least 

 part of the RNA and not RNA quantity in itself is a decisive factor in this process. 

 Borsook (1956a, p. 56) concludes that in rabbit reticulocytes rapid protein 

 synthesis coincides with a low RNA content and turnover of only a small RNA 

 fraction. 



In the course of development the survival of certain embryonic cells soon be- 

 comes dependent on numerous extracellular factors. To cite one example, inner- 

 vation becomes an essential condition for regeneration (Singer, 1952; Singer, 

 Flinker and Sidman, 1956) and for the maintenance, growth and differentiation 

 of muscle tissue (Eastlick, 1943) and in turn the development and growth of the 

 nervous system become dependent on its interaction with peripheral organ 

 rudiments^. The actual isolation from a sarcoma and from snake venom^ of an 

 interesting protein fraction which stimulates proliferation and neuron growth of 

 sensory and sympathetic ganglia has been accomplished (Cohen, Levi-Montalcini 

 and Hamburger, 1954). 



^ The factors which give these results a certain degree of uncertainty are the following : 

 For each experimental condition only a single measurement was made for each time 

 period. The two animals used for the entire experimental series showed large differences 

 in the output of erythrocytes and a wide variation was observed in the ratios of glycine 

 incorporation in the hemin and globin where a constant ratio should have been expected 

 (Exp. II, aandb). Finally, it is not evidentjust why the total amount of glycine incorporated 

 into hemin after 38 h. is smaller than after 6 h. If this would be due to a rapid turnover, and 

 equilibration with decreasing activities of the glycine pool, the rate of hemin synthesis 

 would have to be corrected by this factor. 



^ Work on this phase of development has been reviewed by Hamburger (1955). 

 2 Personal communication. 



