90 PRINCIPLES OF EMBRYOLOGY 



that the internal regions of the egg can be shifted about by centrifugation 

 without producing any profound result, so that the controlling gradients 

 must be in the stiff ectoplasmic layer which is not displaced by centri- 

 fugation. 



Finally, to the question of how the gradients work, we can offer at 

 least the beginning of an answer. Although moderate centrifugation, 

 sufficiently strong to cause considerable stratification of the cytoplasm, 

 has no great effect on development, the situation is rather different follow- 

 ing very intense centrifugation in a modem ultra-centrifuge. Using forces 

 of the order of 45,000 g, on the unfertilised egg. Pease (1939) was able to 

 throw down to the centripetal end a mass of small mitochondria-like 

 granules. He found that if, owing to the orientation of the egg in centri- 

 fuge, these had been collected at the animal pole, the vegetative pole 

 which lacked them was unable to gastrulate; and so, mutatis mutandis, 

 was the animal pole unable to develop if all the granules had been forced 

 to the vegetative end. He suggested that these granules are the im- 

 mediate agents of differentiation. 



At the present time, a great deal of attention is being paid to the be- 

 haviour of various types of cell granules in developing echinoderms. 

 They can be roughly classified into 'mitochondria', which are larger and 

 become sedimented at relatively low speeds of the centrifuge (giving 

 about 16,000 g), and 'microsomes', which are only sedimented at the 

 highest speeds (giving about 100,000 g). The mitochondria can be 

 detected by normal cytological techniques witliiti the cells of the embryo. 

 It has been found (Gustafson and Lenique 1952, Lenique, Horstadius and 

 Gustafson 1953) that in early stages they are distributed in a gradient, de- 

 creasing in concentration from the animal to the vegetable pole (Fig. 5.6). 

 In isolated animal or vegetative halves, and in halves which have been 

 'animalised' or 'vegetalised', the gradients become altered in a manner 

 exactly parallel to that of the basic animal and vegetative gradients, or 

 the gradients in dye reduction mentioned above. This makes it most 

 probable, then, that the mitochondria are rather directly concerned with 

 the fundamental developmental processes. Gustafson (1953, 1954) has 

 made extensive studies on their metabohsm, and suggests that they are 

 connected with the synthesis of the fibrous proteins of the apical tuft, 

 which is the most characteristic animal organ. 



The mitochondria can be formed anew during development. Harvey 

 (1946) centrifuged echinoderm eggs in a medium which had a gradient of 

 specific gravity, and showed that under these circumstances, the egg may 

 be split into several fragments, each containing the constituents of a 

 particular density. Fragments made in this way may develop normally 



