OF THE LYMPHATIC SYSTEM OF THE CHICK. 453 



Although the origin of this primary lymphatic plexus has not yet been deter- 

 mined for birds and mammals, studies have been made on what appear to be the 

 first stages of lymphatic development in amphibians. Fedorowicz (1913) studied 

 the origin of the posterior lymph-heart in frogs, and described the first lymphatic 

 vessels as solid strands of cells derived from the endothelium of the lateral caudal 

 vein. These subsequently acquire lumina, unite with one another, and later 

 coalesce to form the lymph-heart. 



Kampmeier (1915) studied the early stages in the development of the anterior 

 lymph sinus in Bufo. His discovery that the endothelial cells in this space retain 

 the yolk-granules longer than do the connective-tissue cells made possible a careful 

 cytological study of the early stages with high powers of the microscope. Kamp- 

 meier came to the conclusion that the first lymphatic vessels are derived from the 

 veins by a process of outgrowth in a number of places. These outgrowths, many of 

 which are at first solid, unite with one another to form the plexus which precedes the 

 formation of the lymph-sinus. 



Beginning with this work in 1912, the authors have spent several years in a 

 careful study, by various methods, of these early lymphatic capillaries in the region 

 of the posterior lymph-heart of the chick in an effort to discover their origin. 



METHOD. 



Our method of studying living chicks has previously been described (Clark 

 and Clark, 1912, 1914). The essentials are a binocular microscope inclosed in a 

 warm chamber kept at incubator temperature, Ringer's solution for keeping the 

 chick moist, and a bright light for direct illumination (sunlight or a desk-arc). A 

 large window was made in the egg-shell, and the amnion opened and pulled aside. 

 In this way chicks were kept alive and active for 7 or 8 hours. 



For the lymphatic injections we used India ink, diluted to one-half with tap 

 water, and fine glass cannulae (10 to 15 /* at the tip) attached to a rubber tube. 

 For double injections we used various materials, India ink for the lymphatics and 

 Berlin blue with 5 per cent gelatin for the blood-vessels; or India ink in the blood- 

 vessels and Berlin blue in the lymphatics. We also injected lymphatics with silver 

 nitrate (0.5 per cent) and the blood-vessels with India ink. Such injected speci- 

 mens were fixed in Carnoy's fluid, dehydrated in absolute alcohol, and cleared in 

 benzol and oil of wintergreen. Or, if we desired to section such embryos, they were 

 fixed in Bouin's fluid. 



For our studies of sections the blood-vessels were injected completely with 

 India ink. Good injections were obtained either through the allantoic artery or 

 through one of the vitelline veins. In either case the heart was allowed to pump the 

 injection mass around through the body. The umbilical cord was tied and the 

 embryo taken from the yolk and dropped into the fixing solution. For this purpose 

 we obtained the best results by the use of McClung's modification of Bouin's fluid: 

 Saturated aqueous solution of picric acid, 75 per cent; formalin (40%), 20 per cent; 

 glacial acetic, 5 per cent. 



The embryos were carefully dehydrated to avoid shrinkage, and then embedded 

 in paraffin. In some cases cross-sections of the tail region were made, but in most 



