A simple method was devised for the maintenance of the chorioallantoic membrane (CAM) of chick embryos in organ culture. Explants of CAM survived for up to 5 days in this system and retained the characteristic three-layered morphology (ectoderm, mesoderm, and endoderm). Induction of the CAM calcium-binding protein (CaBP) by effectors of calcium metabolism was studied in these organ cultures. Vitamin K was found to elicit a seven- to eightfold increase in CaBP, whereas no increase in CaBP activity occurred on supplementation with vitamin A, parathyroid hormone, an analogue of vitamin D, vitamin D and its hydroxylated metabolites, or with elevated calcium levels. The vitamin K-mediated induction of CaBP was dose-dependent, inhibited by the vitamin K antagonists warfarin and dicoumarol, selective for vitamin K5, and maximal at the developmental stage (13-15 days of incubation) corresponding to the onset of calcium transport by the CAM in vivo. CaBP levels increased after 60-70 h in cultures of 13-15 day CAM supplemented with vitamin K and reached maximal levels around 80-90 h of culture. The CAM ectoderm underwent extensive proliferation and often assumed a villuslike morphology in the vitamin K cultures.
The preparation of a specific antiserum (anti-CaBP) against the calcium-binding protein (CaBP) of the chorioallantoic membrane (CAM) is described. The anti-CaBP appeared to be specific for the CaBP by immunodiffusion and immunoelectrophoresis. Application of the anti-CaBP in immunofluorescence histochemistry revealed that the CaBP is present in the CAM only at developmental ages corresponding with the expression of the calcium transport function of the membrane. Furthermore, the CaBP is localized to the ectoderm of the CAM, appears to be exposed to the entire external surface of the ectoderm, and can be shown to be associated with cells enzymatically dissociated from the CAM. These results are consistent with a functional role of the CaBP in the CAM calcium transport process.