Development 105, 147-153 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 147 Presence of basic fibroblast growth factor in the early Xenopus embryo J. M. W. SLACK and H. V. ISAACS Imperial Cancer Research Fund, Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX13PS Summary Mesoderm-inducing activity can be extracted from Xenopus embryos, eggs or whole ovary. It binds to heparin and can be neutralized by heparin or anti-bFGF but not by anti-TGF/?. Two molecular forms can be identified by Western blotting and have molecular weights of about 19 and 14K. The content in embryos is about 7 units g~' (approximately 7 ng ml~*) which would be sufficient for it to be acting as an endogenous inducer of ventral mesoderm. Attempts to detect TGF/5-like inducing factors in embryos were not successful. Key words: Xenopus oocyte, egg, blastula, heparin-binding growth factors, fibroblast growth factor, transforming growth factor beta, mesoderm-including factors. Introduction Much interest has been aroused recently by the possi- bility of identifying the morphogens responsible for mesoderm induction in the early amphibian embryo. This is the process by which the mesoderm is induced from the animal hemisphere of the blastula by signals from the vegetal region (Nieuwkoop, 1969; Dale et al. 1985; Gurdon et al. 1985; Jones & Woodland, 1987). Recently we reported that a small group of heparin- binding growth factors (HBGFs) are active as meso- derm-inducing agents and the relatively high degree of specificity suggested to us that at least the ventral mesoderm induction within the embryo was mediated by an HBGF (Slack et al. 1987). However, it has now been found that another type of growth factor, TGF/J-2, is active (Rosa et al. 1988) and a mesoderm-inducing factor recently purified from a Xenopus cell line is also thought to belong to the TGF/3 family (Smith, 1987; Smith et al. 1988). So which, if any, of these factors is really involved in mesoderm induction in the early embryo? A clue was provided by Kimelman & Kirschner (1987) who detected an mRNA of the bFGF type in Xenopus embryos. We now show that a meso- derm-inducing factor can be isolated from Xenopus blastulae. It is similar to bFGF by biochemical and immunological criteria and has a similar mesoderm- inducing activity in vitro. The quantity present is small but sufficient for it to be responsible for induction in vivo. This is consistent with, although does not yet finally prove, the involvement of bFGF as a morphogen in mesoderm induction. Materials and methods Extraction of HBGF from Xenopus material Eggs and embryos were dejellied with 2% cysteine hydro- chloride pH7-9 and stored at -70°C before use, as were whole ovaries from adult females. The final protocol adopted was as follows: 25-100 g of ovary, unfertilized eggs or em- bryos were homogenized in 2-3 vols of 0-15M-ammonium sulphate containing lmM-PMSF and 100 nin-pepstatin A. Homogenization was by Ultra turrax blender for 3x1 min at maximum speed, the temperature being kept below 10°C with ice. All subsequent steps up to the Amicon concentration were carried out at cold room temperature. The pH was adjusted to 4-5 with acetic acid and the homogenate was stirred for 1 h on ice. It was then centrifuged at 150000g for one hour and lipid was removed by straining through nylon mesh. The supernatant was neutralized with ammonia and fractionated by adding, successively, 0-24 g ml" 1 (40%) and 0'2gml"' (70%) solid ammonium sulphate followed by centrifugation at 12000g for 10min. The 40-70% cut was dialysed overnight against 0-6M-NaCl, 20mM-Tris pH7-0 and then applied to a 2 ml column of heparin Sepharose (Sigma or Pharmacia) equilibrated in the same buffer. The column was washed in the same buffer and eluted with 2M-NaCl, 20 HIM- Tris-HCl pH7-0. Ovalbumin (Sigma, mol. wt std) was added as carrier and the product was concentrated to 1 ml with an Amicon ultrafiltration cell and YM10 membrane. This ma- terial is referred to below as the 'heparin-bound fraction'. Earlier preparations differed in that the initial homogenate was centrifuged less vigorously (25 OOOg, 30 mins), a 40-80 % ammonium sulphate precipitate was taken, and also that a CM-Sephadex column at pH6 was employed before the heparin column. Western blots The samples were separated using 15 % SDS-polyacrylamide gels and transferred to nitrocellulose using standard tech-