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ORGANIZATION AND CELL-LINEAGE ОҒ ASCIDIAN EGG. 47 
the mid-line and right at the middle of the crescent, of which they form an 
extremely small part. They are formed by the aggregation at this point of a little 
clear protoplasm which first appeared at the time of fertilization as a clear area 
around the spermatozoon, and which afterwards lies at the middle of the crescent 
(figs. 173, 175,176). In Cynthia this area of clear protoplasm does not usually 
take the form of the deeply staining bosses or caps before the 16-cell stage (figs. 
113, 115), though these may sometimes appear, as they do in Crona, at the 8-cell 
stage. Although they arise from the surface of the crescent they contain no yellow 
pigment, and in the living egg this small spot of protoplasm and the cells to which 
it gives rise are almost perfectly transparent and are therefore difficult to see. In 
stained preparations they always stain deeply and thus form an excellent land- 
mark (figs. 116-120, et seg.). 
Chabry and Castle have called particular attention to these prominences of 
clear protoplasm which are found at the posterior pole of the egg, and Castle traces 
them back to the 2-cell stage, and gives good reason for believing that this area 
of clear protoplasm marks the point of entrance of the spermatozoon, and was 
caused by it. I entirely agree with Castle that this aggregation of clear protoplasm 
is caused by the entering spermatozoon, since I have seen it surrounding the sper- 
matozoon immediately after its entrance (fig. 173); but it can scarcely be said to 
mark the point of entrance, as it does not remain stationary but moves with the 
crescent from a point near the vegetal pole to one near the equator on the posterior 
side of the egg. So far as I am able to determine from a study of Castle's figures 
and description, the area of finely granular protoplasm, which he represents in his 
figures 17, 45, 46 and 47, is the middle portion of the crescent. The large area of 
clear protoplasm represented in each of these figures and marked x gives rise to 
the middle cells of the crescent (С°, D^? of his fig. 49), therefore the small pos- 
terior mesenchyme cells, C^? and D?? of later stages, can represent but a small part 
of the area marked x in the earlier stages. Тһе earliest stage in which Castle 
represents the substance of these future mesenchyme cells is at the posterior pole 
of the cells C^? and D9? in his figure 51. I conclude, therefore, that he observed 
the middle portion of the crescent (x of his figures) in the earliest stages of the 
development, but that he did not recognize the substance of the small mesenchyme 
cells as distinguished from the substance x (crescent substance) before the 24-сеП 
stage (his fig. 51). 
All students of ascidian embryology agree that the first plane of cleavage is 
median in position, the second transverse, and the third horizontal or coronal, but 
beyond this there are few agreements among them, as has been pointed out. In 
the matter of the relations of these cleavage planes to the germ layers there are as 
many opinions as there are concerning the orientation of the egg. Van Beneden 
and Julin (1884) maintained that the four ventral cells of the 8-cell stage are 
purely ectodermal, but that the four dorsal cells are still * mixed," each of them 
containing ectoderm and endoderm, while not until the 44-cell stage is the separa- 
tion of ectoderm and endoderm in these dorsal cells completed. Seeliger (1885) held 
