Studies on Pattern Formation in Chick Limb Bud.

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1. Growth factor of ZPA.(1) By the use of organ culture of chick limb bud fragments, the presence of growth factor(s) in the posterior region including ZPA and the responsiveness of the anterior region to the factor have been demonstrated.(2) The limb bud was divided into four regions along the anteroposterior axis, and dissociated cells of each region were cocultured with the fragments of the posterior region. The proliferation of the cells from the anterior region was promoted by the coculture, and a gradient of responsiveness to the growth promoting factor was found. No promotion was observed when cultured with anterior fragments.(3) The presence of the gradient of responsiveness was examined on some known growth factors. Fibroblast growth factor(FGF) showed similar pattern of growth promotion, suggesting that ZPA produces and releases FGF-like growth factor.(4) Growth promotion effect of retinoic acid, a canidate of morphogen in limb bud morphogenesis, was examined. In retina-conditoned medium, the retinoic acid at 10-100ng/ml promoted the proliferation and cartilage nodule formation of distal mesodermal cells.2. Limb bud specific protein.Using the method of two dimensional electrophoresis and silver staining. a limb specific protein, L-1, has been found. The study on the distribution of the protein in various limb bud region is now in progress.3. Limb bud region specific antigen, AV-1.The position specific antigen, AV-1, was found to be a glycoprotein with a molecular weight of 135kD.