216 OEIGIN OF BLOOD-VESSELS IN BLASTODERM OF CHICK. 



1868, are based upon studies of living chicks immediately after removing them 

 from the shell; while Duval in his atlas, published in 1889, called attention to the 

 advantage of studying the beating of the heart in living chicks in a watch crystal. 

 Indeed this advantage has been quite apparent to the whole group of embryologists 

 using the same material. As a result of the development of different artificial solu- 

 tions upon which the method of tissue culture depends, there has naturally followed 

 a marked increase in the studies of living embryos of the higher forms; for example, 

 the series of observations made by E. R. and E. L. Clark (1912, 1915) on the super- 

 ficial lymphatics in chicks held in the shell and kept alive by Rusch solution, and 

 the demonstration by Brachet (1913) that a mammalian egg could be kept alive 

 for at least 48 hours in blood-plasma. These studies, though depending upon the 

 development of the media which are used in tissue-culture, are not specifically 

 tissue-cultures, for by that term is meant the growth of tissues on a coverslip in 

 artificial media, so arranged that the development of their cells can be watched 

 under the microscope. 



The first direct application of the method of tissue-culture to the entire blas- 

 toderm of the chick that is, the study of the blastoderm on a coverslip so that its 

 cells could be clearly followed was made by McWhorter and Whipple in 1912. 

 These investigators followed the technique of Burrows and Carrel, developed from 

 the work of Harrison, using clotted chicken-plasma in which to grow the specimens. 

 In this medium they kept chicks under observation for a considerable period of 

 time namely, from the stage of about 2 or 3 somites up to the stage of 17 or 18 

 somites. In my experiments I have followed the method of W. H. and M. R. 

 Lewis, using Locke-Lewis solution instead of the clotted plasma. This technique 

 is much more simple and, I believe, offers many advantages, in spite of the fact that 

 the embryos do not live nearly as long. I have made no attempt to determine the 

 possible duration of life of the specimens in the media, but have watched them on 

 an average of 4 to 5 hours, during which time the maximum number of new somites 

 is 3, with an average of 2. The greater ease of the method renders possible much 

 more extensive observations. My series numbers 266. 



With this method of studying the blastoderm of the chick, it is possible to trace 

 the formation of blood-vessels back to their very beginning and to observe them 

 developing by differentiation from mesoderm to a new type of cell, the angioblast. 

 These angioblasts form solid clumps, then bands or cords of cells, which soon unite 

 in a plexus by the well-known method of sprouting. The central part of the sub- 

 stance, either of the isolated clumps or of the plexus of angioblasts, then liquefies, 

 leaving the rim to form the endothelial wall of the vessel. In this liquefaction both 

 cytoplasm and nuclei are involved, and the resulting fluid constitutes the first blood- 

 plasma. Moreover, angioblasts not only give rise to endothelium and blood-plasma, 

 but also produce the red blood-corpuscles. Thus in the chick angioblasts are cells 

 which differentiate from mesoderm and form endothelium, blood-plasma, and red 

 blood-cells. 



That angioblasts differentiate in the area vasculosa of the chick has long been 

 known ; indeed, the idea is involved in our knowledge of the origin of the so-called 



