The long-term objective of the proposed research is to establish the primordial locations and relationships of the preheart and prelung cells, compare them to their intermediate and final positions, and study heart- and lung-specific molecules and morphogens.
Specific aims of the study include concisely locating the preheart, prelung and heart-specific portions of the neural crest in the epiblast layer between the early short streak and definitive streak stages, exploring the capabilities of non-heart and non-lung forming cells when transplanted into prospective heart and lung forming areas, and conversely, the capabilities of preheart and prelung cells to differentiate into other organs when transplanted into areas of the embryo not destined for heart and lung. Another specific aim is to trace the contributions of right and left heart-forming regions during the developmental period which is characterized by looping and twisting of the primitive heart, in order to better define the formation of the chambers, conduction system and valve apparati. These research endeavors are now possible only because of recent advances in the methodology of permanently marking embryonic cells in ovo. Fertile quail eggs will be explanted and stained lightly with Nile Blue A for contrast. Grafts containing approximately 1-500 cells will be transferred to the dorsal surface of the recipient chick embryo in ovo and inserted into the epiblast or mesoderm layer. Recipient embryos will be reincubated for 6-10 days, fixed, embedded, serially sectioned and stained with the Fuelgen method. By comparing the original and final locations of the grafts, and by studying the differentiative capabilities of the various cells, a comprehensive account of the early development of the heart and lung will result. In separate experimentation, the lateral and medial margins of the heart forming regions will be """"""""tattoo""""""""-labeled with tritiated thymidine in ovo. Study of radioautographs of embryos reincubated for 2-4 days will result in a better understanding of the development of chambers, valves and conduction tissue, because the permanently marked seams between right and left heart forming regions can be identified, even after the processes of looping and torsion have been completed.