ORIGIN OF BLOOD-VESSELS IN BLASTODERM OF CHICK. 217 



blood-islands dating back to the early embryologists, notably Wolff and Pander. 

 In this study I propose to sharpen the conception of the differentiation of angio- 

 blasts and to limit the term blood-island to those masses of cells which actually 

 produce hemoglobin and become red blood-corpuscles. It will be shown that 

 though angioblasts, immediately after their differentiation, begin to spread by 

 sprouting, the primitive vessels do not invade the embryo in the manner believed 

 by His, but that there is a progressive differentiation of angioblasts in situ, starting 

 in the area opaca of the chick and gradually extending toward the embryo. The 

 heart, most of the dorsal aorta, and even a part of the ventral aorta of the head, 

 can be seen in the living chick to differentiate in situ. Thus these studies in the 

 living blastoderm confirm the experiments of Hahn (1909), in which he destroyed 

 the area vasculosa on one side of a very young blastoderm and subsequently found 

 an aorta on that side. These experiments were confirmed later by McWhorter and 

 Miller (1914) and the same point was brought out by Reagan (1915), who isolated 

 the head-fold of a young embryo and found vessels in the isolated segment. It 

 may thus be considered as settled that blood-vessels arise from cells which differ- 

 entiate from mesoderm, and that these cells (angioblasts) differentiate not only 

 throughout the area vasculosa, but also throughout the body of the embryo itself. 

 The ultimate period at which angioblasts cease to differentiate from mesenchyme 

 must be regarded as still unknown. 



METHODS. 



My early studies were made in the following Locke-Lewis solution: NaCl 

 0.9, KC1 0.042, CaCl 2 0.024, NaHCO 3 0.02, Glucose 0.25 with chicken bouillon. 



It is convenient to make stock solutions of the salts four times the desired 

 strength and mix 25 c.c. of each. To this mixture is added the glucose and 20 c.c. 

 of chicken bouillon. This is made according to the method for media and carefully 

 neutralized with sodium bicarbonate. The bouillon has also 0.50 grams of sodium 

 chloride to each 100 c.c. in order not to dilute the Locke solution too much. The 

 mixture of Locke solution and bouillon (Locke-Lewis solution) is then divided into 

 test tubes and heated in an Arnold sterilizer. It may be put through a Bergfelt 

 filter instead, but this has no advantage except to prevent the solution from becom- 

 ing spoiled during sterilization by the formation of a precipitate, an accident which 

 occasionally happens. 



This Locke-Lewis fluid is, of course, a solution in which the exact amount of 

 salts is unknown, owing to the addition of the bouillon; but since a given solution 

 of bouillon can be kept sterile and made to serve for a very large number of experi- 

 ments, there is a possibility of making certain experiments with a solution which 

 varies only in the known amount of salts added. This solution has been shown by 

 W. H. and M. R. Lewis to be excellent for the growth of tissues of chicks that have 

 been incubated for from 6 to 12 days, but in studying the entire blastoderm in this 

 fluid I noted that in specimens of the latter half of the second day, by which time 

 circulation had been established, nearly all of the hemoglobin was laked out of the 

 corpuscles. Following the studies of Bialaszewicz (1912), who showed that an 



