Identification of a novel growth factor with transforming activity secreted by individual chick embryos

Previously we have developed a microassay for anchorage independent growth (AIG) of fibroblasts in soft agar, which can detect very small quantities of transforming growth factors (TGFs). Using this assay, we have shown that small pieces of dissected chick embryo tissue will stimulate AIG of both NR6 and NRK 49f cells, and that this property can be used to map production of growth factors with transforming activity in individual early embryos. We now show that this activity can be transferred to conditioned medium (CM) prepared using chick embryo tissue. Using two cell lines with differential responsiveness to TGFs, and by coincubating normal and heat-treated CM with trypsin, Con-A and neutralising antibodies, we show that CM contains at least two different growth factors with transforming activity. One of these is heat-stable, and stimulates colony formation in NRK 49f cells in the presence of EGF, but not in its absence. This activity corresponds to a TGF beta-like molecule. The other component is a heat-labile glycoprotein, which has TGF alpha-like properties, but does not appear to behave like known TGFs with these properties. It therefore appears to be a novel growth factor. Both activities are present from the intermediate primitive streak stage of development.