204 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 



then, it is doubly so now, for the literature of the subject has since that 

 time multiplied many fold. Nevertheless there still remain many un- 

 settled questions regarding the embryology of the Tunicates. Concern- 

 ing so fundamental a point as the derivation of the primary germ layers 

 in the embryo, quite contradictor}^ opinions have been expressed within 

 the last ten years by observers of world-wide reputation. 



I undertook the inquiry, the results of which are recorded in the fol- 

 lowing pages, in the hope of being able to throw light on this disputed 

 question by the study of other forms than those which had been most 

 carefully examined, and by the application of new methods to the prob- 

 lem. A short experience convinced me that the only method which could 

 yield positive conclusions was that of cell lineage, a method which has 

 been applied so successfully to the study of annelid and molluscan em- 

 bryology by a number of observers, and had already been employed to a 

 limited extent in the study of ascidian embryology by Van Beneden et 

 Julin ('84), Seeliger ('85), and Chabry ('87). 



It soon became clear to me that some of the conflicting statements 

 made by my predecessors arose from errors on their part due to incor- 

 rect orientation of certain stages. The nature of these errors I have 

 fully explained in a preliminary communication (Castle, '94). A further 

 study of the embryonic history, cell by cell, through the periods of cleav- 

 age and gastrulation, and even down to the differentiation of the several 

 larval organs, has led me to conclusions somewhat at variance with those 

 of earlier investigators regarding the origin of the primary germ layers 

 and the organs derived from them. One of the most important of these 

 conclusions is that the mesoderm of Ascidians — and probably also that 

 of Amphioxus and the Vertebrates — is derived in part from the primary 

 entoderm and in part from the primary ectoderm. The grounds on 

 which this conclusion rests are set forth in the later portions of this 

 paper ; in the earlier part of the paper I have recorded some observa- 

 tions on the maturation and fertilization of the ascidian egg. 



It gives me pleasure to acknowledge in this place my very great obli- 

 gations to Professor E. L. Mark for direction and kindly criticism of my 

 entire work. My best thanks are also due to Dr. Alexander Agassiz, in 

 whose laboratory at Newport the material for my studies was chiefly 

 collected, and to Colonel Marshall McDonald for numerous courtesies 

 extended to me at the United States Fish Commission Station at 

 Wood's Holl. 



