Histochemical Localization of Enzymes in the Embryonic Chick Liver

Oxidative and hydrolytic enzymes were localized histochemically in the developing chick liver and in liver grown as chorioallantoic (CAM) transplants or as explants in organ culture. In the i n situ liver, parenchymal cells exhibited an increase in succinate (SDH) and a glycerophosphate (GDH) dehydrogenase activity and a decrease in NADP diaphorase (TPND) during development. NAD diaphorase (DPND) and nonspecific esterase (NSE) activity remained constantly high throughout the period studied. Within the stromal cells, DPND and NSE activity decreased and SDH, GDH and alkaline phosphatase (ALP) increased. The ALP activity of the sinusoidal cells appeared to increase coincident with a redistribution of TPND within the cytoplasm of the parenchymal cells. In CAM transplants, the enzyme activity of all tissues was nearly the same as the five day liver even though the total age of the tissues was 15 days. Liver explants exhibited enzyme activity similar in part to both the 5 and 15 day liver. In both explants and transplants, ALP activity was highest in areas where the parenchymal-sinusoidal relationship was maintained and absent in areas where the parenchyma and sinusoidal cells were not in contact. In a recent report (Conklin, '64), the cytochemical localization of lipid, glycogen and the enzyme, lactate dehydrogenase (LDH), in the chick hepatic epithelium was described. It was noted that a correlation existed between the glycogen and lipid content of the hepatic cells and the ontogenesis of certain of the LDH isozymes (Nebel and Conklin, '64). Also noted was an alteration in LDH isozymes of liver which had been transplanted to the chorioallantoic membrane (CAM) of the chick. The study of the embryonic liver has been extended to include additional enzymes, both oxidative and hydrolytic, in order to further characterize the developing hepatic parenchyma. In addition, a comparison has been made between enzyme distribution in epithelial and connective tissue cells. Finally, since preliminary observations indicated a change in the LDH activity of liver transplants, enzyme activity was also studied in liver grown both in nitro, as organ explants, and in vivo, as chorioallantoic transplants. MATERIALS AND METHODS The material consisted of excised samples of liver from 20 five day and 55 15 J. EXP. ZOOL., 161: 251-270. day (Hamburger-Hamilton, '51) white leghorn chick embryos and livers grown as transplants (45) and explants (20). In removing samples for enzyme demonstration or transplantation samples were taken at random from the liver. Since samples of the same age or subjected to the same treatment exhibited similar enzyme activity it is probable that regional variation within the liver was not a significant factor in the results obtained. For transplantation livers were taken from five day donor embryos and grown for ten days in eight day host embryos. The liver of the five day embryo was removed aseptically and placed in sterile, avian saline. It was then cut into small pieces, 1-2 mm in size, which were used as the transplant. Preparation of the host embryo consisted of cutting a 3 X 5 mm rectangle in the egg shell with a dental saw, removing the shell membrane and gently (with a sterile needle) breaking a small vessel in the chorioallantoic membrane (CAM) so that a drop or two of blood leaked onto the surface of the CAM. When leakage was judged to be excessive IResearch supported by grant HD 00557 from NICHD, National Institutes of Health and grant ACS IN 40-D from the University of Michigan Cancer Research Institute.