The technique for whole embryo culture was based on the methods described by New [31]. The gravid uterus was removed and placed in sterile PBS (0.1 M phosphate buffer containing saline) at 37°C. The embryo in the visceral yolk sac along with a small piece of the ectoplacental cone (hereafter called embryo, unless otherwise stated) was carefully removed from the deciduas tissues and the Reichert's membrane in PBS containing 4% fetal bovine serum (Sigma, St Louise. MO). After removal, three embryos bearing 3-5 somites (E8.25) were incubated in a culture bottle in 20 mL of medium which consisted of 70% immediately centrifuged heat-inactivated rat serum (Harlan Sprague-Dawley, Inc, Indianapolis, IN) and 30% phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 8 mM Na2HPO4, 1.47 mM KH2PO4, 0.9 mM CaCl2, 5.6 mM glucose, 0.33 mM sodium pyruvate, pH7.4) supplemented with 20 units/ml penicillin and 20 units/ml streptomycin (Sigma, St. Louis, MO), and gassed with 5% O2, 5% CO2, and 90% N2 in a rotating culture system (B.T.C. Precision Incubator Unit, B.T.C. engineering, Cambridge, England, 36 rpm) for
